Kinase inhibitors and methods of their use

ABSTRACT

New compounds, compositions and methods of inhibition of Provirus Integration of Maloney Kinase (PIM kinase) activity associated with tumorigenesis in a human or animal subject are provided. In certain embodiments, the compounds and compositions are effective to inhibit the activity of at least one PIM kinase. The new compounds and compositions may be used either alone or in combination with at least one additional agent for the treatment of a serine/threonine kinase- or receptor tyrosine kinase-mediated disorder, such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims the benefit under 35U.S.C. §120 and §121 of U.S. patent application Ser. No. 13/327,358,filed Dec. 15, 2011, now issued as U.S. Pat. No. 8,592,455, which is adivisional application of U.S. Ser. No. 12/584,158, filed Aug. 31, 2009,which issued as U.S. Pat. No. 8,329,732 and which in turn claims thebenefit under 35 U.S.C. §119(e) to U.S. provisional application serialNo. 61/093,666, filed on Sep. 2, 2008, and to U.S. provisionalapplication serial No. 61/225,660, filed Jul. 15, 2009. The entirecontents of each of these prior applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to new compounds and their tautomers andstereoisomers, and pharmaceutically acceptable salts, esters,metabolites or prodrugs thereof, compositions of the new compoundstogether with pharmaceutically acceptable carriers, and uses of the newcompounds, either alone or in combination with at least one additionaltherapeutic agent, in the prophylaxis or treatment of cancer.

BACKGROUND

Infection with the Maloney retrovirus and genome integration in the hostcell genome results in development of lymphomas in mice. ProvirusIntegration of Maloney Kinase (PIM-Kinase) was identified as one of thefrequent proto-oncogenes capable of being transcriptionally activated bythis retrovirus integration event (Cuypers H T et al., “Murine leukemiavirus-induced T-cell lymphomagenesis: integration of proviruses in adistinct chromosomal region,” Cell 37(1):141-50 (1984); Selten G, etal., “Proviral activation of the putative oncogene Pim-1 in MuLV inducedT-cell lymphomas” EMBO J 4(7):1793-8 (1985)), thus establishing acorrelation between over-expression of this kinase and its oncogenicpotential. Sequence homology analysis demonstrated that there are 3highly homologous Pim-Kinases (Pim1, 2 & 3), Pim1 being theprotooncogene originally identified by retrovirus integration.Furthermore, transgenic mice over-expressing Pim1 or Pim2 show increasedincidence of T-cell lymphomas (Breuer M et al., “Very high frequency oflymphoma induction by a chemical carcinogen in pim-1 transgenic mice”Nature 340(6228):61-3 (1989)), while over-expression in conjunction withc-myc is associated with incidence of B-cell lymphomas (Verbeek S etal., “Mice bearing the E mu-myc and E mu-pim-1 transgenes developpre-B-cell leukemia prenatally” Mol Cell Biol 11(2):1176-9 (1991)).Thus, these animal models establish a strong correlation between Pimover-expression and oncogenesis in hematopoietic malignancies. Inaddition to these animal models, Pim over-expression has been reportedin many other human malignancies. Pim1, 2 & 3 over-expression isfrequently observed in many hematopoietic malignancies (Amson R et al.,“The human protooncogene product p33pim is expressed during fetalhematopoiesis and in diverse leukemias,” PNAS USA 86(22):8857-61 (1989);Cohen A M et al., “Increased expression of the hPim-2 gene in humanchronic lymphocytic leukemia and non-Hodgkin lymphoma,” Leuk Lymph45(5):951-5 (2004), Huttmann A et al., “Gene expression signaturesseparate B-cell chronic lymphocytic leukaemia prognostic subgroupsdefined by ZAP-70 and CD38 expression status,” Leukemia 20:1774-1782(2006)) and in prostate cancer (Dhanasekaran S M, et al., “Delineationof prognostic biomarkers in prostate cancer,” Nature 412(6849):822-6(2001); Cibull T L, et al., “Overexpression of Pim-1 during progressionof prostatic adenocarcinoma,” J Clin Pathol 59(3):285-8 (2006)), whileoverexpression of Pim3 is frequently observed in hepatocellularcarcinoma (Fujii C, et al., “Aberrant expression of serine/threoninekinase Pim-3 in hepatocellular carcinoma development and its role in theproliferation of human hepatoma cell lines,” Int J Cancer 114:209-218(2005)) and pancreatic cancer (Li Y Y et al., “Pim-3, a proto-oncogenewith serine/threonine kinase activity, is aberrantly expressed in humanpancreatic cancer and phosphorylates bad to block bad-mediated apoptosisin human pancreatic cancer cell lines,” Cancer Res 66(13):6741-7(2006)).

Pim1, 2 & 3 are Serine/Threonine kinases that normally function insurvival and proliferation of hematopoietic cells in response to growthfactors and cytokines. Cytokines signaling through the Jak/Stat pathwayleads to activation of transcription of the Pim genes and synthesis ofthe proteins. No further post-translational modifications are requiredfor the Kinase Pim activity. Thus, signaling down stream is primarilycontrolled at the transcriptional/translational and protein turnoverlevel. Substrates for Pim kinases include regulators of apoptosis suchas the Bcl-2 family member BAD (Aho T et al., “Pim-1 kinase promotesinactivation of the pro-apoptotic Bad protein by phosphorylating it onthe Serl 12 gatekeeper site: FEBS Letters 571: 43-49 (2004)), cell cycleregulators such as p21^(WFA1/CIP1) (Wang Z, et al., “Phosphorylation ofthe cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase,” Biochem BiophysActa 1593:45-55 (2002)), CDC25A (1999), C-TAK (Bachmann M et al., “TheOncogenic Serine/Threonine Kinase Pim-1 Phosphorylates and Inhibits theActivity of Cdc25C-associated Kinase 1 (C-TAK1). A novel role for Pim-1at the G2/M cell cycle checkpoint,” J Biol Chem 179:48319-48328 (2004))and NuMA (Bhattacharya N, et al., “Pim-1 associates with proteincomplexes necessary for mitosis,” Chromosoma 111(2):80-95 (2002)) andthe protein synthesis regulator 4EBP1 (Hammerman P S et al., “Pim andAkt oncogenes are independent regulators of hematopoietic cell growthand survival,” Blood 105(11):4477-83 (2005)). The effects of Pim(s) inthese regulators are consistent with a role in protection from apoptosisand promotion of cell proliferation and growth. Thus, over-expression ofPim(s) in cancer is thought to play a role in promoting survival andproliferation of cancer cells and, therefore, their inhibitions shouldbe an effective way of treating cancers on which they areover-expressed. In fact several reports indicate that knocking downexpression of Pim(s) with siRNA results in inhibition of proliferationand cell death (Dai J M, et al., “Antisense oligodeoxynucleotidestargeting the serine/threonine kinase Pim-2 inhibited proliferation ofDU-145 cells,” Acta Pharmacol Sin 26(3):364-8 (2005); Fujii et al. 2005;Li et al. 2006). Furthermore, mutational activation of several well knowoncogenes in hematopoietic malignancies are thought exert its effects atleast in part through Pim(s). For example, targeted down regulation ofpim expression impairs survival of hematopoietic cells transformed byFlt3 and BCR/ABL (Adam et al. 2006). Thus, inhibitors to Pim1, 2 &3would be useful in the treatment of these malignancies. In addition to apotential role in cancer treatment and myeloproliferative diseases, suchinhibitor could be useful to control expansion of immune cells in otherpathologic condition such as autoimmune diseases, allergic reactions andin organ transplantation rejection syndromes. This notion is supportedby the findings that differentiation of Th1 Helper T-cells by IL-12 andIFN-α results in induction of expression of both Pim1&2 (Aho T et al.,“Expression of human Pim family genes is selectively up-regulated bycytokines promoting T helper type 1, but not T helper type 2, celldifferentiation,” Immunology 116: 82-88 (2005)). Moreover, Pim(s)expression is inhibited in both cell types by the immunosuppressiveTGF-β (Aho et al. 2005). These results suggest that Pim kinases areinvolved in the early differentiation process of Helper T-cells, whichcoordinate the immunological responses in autoimmune diseases, allergicreaction and tissue transplant rejection.

A continuing need exists for compounds that inhibit the proliferation ofcapillaries, inhibit the growth of tumors, treat cancer, modulate cellcycle arrest, and/or inhibit molecules such as Pim1, Pim2 and Pim3, andpharmaceutical formulations and medicaments that contain such compounds.A need also exists for methods of administering such compounds,pharmaceutical formulations, and medicaments to patients or subjects inneed thereof.

SUMMARY OF INVENTION

New compounds, and their stereoisomers, tautomers and pharmaceuticallyacceptable salts, are provided of the Formula I

-   -   wherein,    -   X₁, X₂, X₃ and X₄ are independently selected from CR₂ and N;        provided that at least one but not more than two of X₁, X₂, X₃        and X₄ are N;    -   Y is selected from a group consisting of cycloalkyl, partially        unsaturated cycloalkyl, and heterocycloalkyl, wherein each        member of said group may be substituted with up to four        substituents;    -   Z₂ and Z₃ are independently selected from CR₁₂ and N; provided        that not more than one of Z₂ and Z₃ can be N;    -   R₁ is selected from the group consisting of hydrogen, —NHR₃        halo, hydroxyl, alkyl, cyano, and nitro;    -   R₂ and R₁₂ independently at each occurrence are selected from        the group consisting of hydrogen, halo, hydroxyl, nitro, cyano,        SO₃H and substituted or unsubstituted alkyl, alkenyl, alkynyl,        alkoxy, amino, cycloalkyl, hetero cycloalkyl, and partially        saturated cycloalkyl;    -   R₃ is selected from the group consisting of hydrogen, —CO—R₄ and        substituted or unsubstituted alkyl, cycloalkyl, heterocyclyl,        aryl and heteroaryl;    -   R₄ is selected from the group consisting of alkyl, substituted        alkyl, alkoxy, substituted alkoxy, amino, substituted amino, and        alkylamino; and    -   R₅ represents a group selected from substituted or unsubstituted        aryl, C₃-C₇ cycloalkyl, heteroaryl, partially unsaturated        cycloalkyl and alkyl, wherein each said substituted R₅ group may        be substituted with up to four substituents selected from halo,        cyano, amino, C₁₋₄ alkyl, C₃₋₆ cycloalkyl, alkoxy, nitro,        carboxy, carbonyl, carboalkoxy, aminocarboxy, substituted        aminocarbonyl, aminosulfonyl, substituted aminosulfonyl and        alkoxyalkyl.

In some embodiments, new compounds of Formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein X₂ is N and X₁, X₃ and X₄ are CR₂.

In some embodiments, new compounds of Formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₂ is selected from hydrogen, methyl, ethyl, halo, cyano.

In some embodiments, compounds of Formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein Z₂ and Z₃ are CR₁₂.

In some embodiments, compounds of Formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₁₂ is selected from hydrogen, halo, methyl, ethyl and cyano.

In other embodiments, new compounds, and their stereoisomers, tautomersand pharmaceutically acceptable salts, are provided of the Formula II

-   -   wherein,    -   Y is selected from a group consisting of cyclohexyl, partially        unsaturated cyclohexyl, and heterocyclo-C₅-alkyl, wherein each        member of said group may be substituted with up to four        substituents;    -   R₁ is selected from the group consisting of hydrogen, —NHR₃        halo, hydroxyl, alkyl, C₃₋₄ cycloalkyl, cyano, and nitro;    -   R₁₂ independently at each occurrence is selected from the group        consisting of hydrogen, halo, hydroxyl, amino, nitro, cyano,        SO₃H and substituted or unsubstituted alkyl, alkenyl, alkynyl,        alkoxy, amino, cycloalkyl, hetero cycloalkyl, and partially        saturated cycloalkyl;    -   R₃ is selected from the group consisting of hydrogen, —CO—R₄ and        substituted or unsubstituted alkyl, cycloalkyl, heterocyclyl,        aryl and heteroaryl;    -   R₄ is selected from the group consisting of alkyl, substituted        alkyl, alkoxy, substituted alkoxy, amino, substituted amino, and        alkylamino; and    -   R₅ represents a group selected from hydrogen and substituted or        unsubstituted alkyl, C₆-cycloalkyl, aryl and heteroaryl, wherein        each said substituted R₅ group may be substituted with up to        four substituents selected from halo, cyano, amino, C₁₋₄ alkyl,        C₃₋₆ cycloalkyl, alkoxy, nitro, carboxy, carbonyl, carboalkoxy,        aminocarboxy, substituted aminocarbonyl, aminosulfonyl,        substituted aminosulfonyl and alkoxyalkyl.

In some embodiments, compounds of Formulas I or II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein Y is selected from a group consisting of substituted orunsubstituted cycloalkyl, cycloalkenyl, piperidinyl and piperazinyl,wherein each member of said group is substituted with up to foursubstituents. In some embodiments, Y is substituted with up to foursubstituents selected from, cyano, nitro, halo, hydroxyl, amino, alkoxy,substituted amino, C₁₋₄ alkyl, C₁₋₄ halo alkyl and C₃₋₄ cycloalkyl. Inyet other embodiments, Y is substituted with up to four substituentsselected from methyl, propyl, i-propyl, ethyl, hydroxyl, amino, halo,monohalo C₁₋₃ alkyl, trihalo C₁₋₃ alkyl and dihalo C₁₋₃ alkyl.

In some embodiments, new compounds of Formula II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein Y is selected from a group consisting of substituted orunsubstituted cyclohexyl, cyclohexynyl, and piperidinyl, wherein eachmember of said group is substituted with up to four substituents.

In some embodiments, new compounds of Formula II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein Y is substituted with up to four substituents independentlyselected from hydrogen, cyano, nitro, halo, hydroxyl, amino, alkoxy,substituted amino, C₁₋₄ alkyl, C₁₋₄ halo alkyl and C₃₋₄ cycloalkyl. Insome embodiments, the substituents are independently selected frommethyl, propyl, i-propyl, ethyl, hydroxyl, amino, halo, monohalo C₁₋₃alkyl, trihalo C₁₋₃ alkyl and dihalo C₁₋₃ alkyl.

In some embodiments, compounds of Formula II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₁₂ is selected from hydrogen, halo, methyl, ethyl and cyano.

In some embodiments, compounds of Formulas I or II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein

Y is selected from the group consisting of substituted or unsubstitutedcyclohexyl, cyclohexenyl, piperidinyl, piperazinyl, wherein said the Ygroup may be substituted with up to three substituents selected frommethyl, ethyl, hydroxyl, amino, and methoxy; R₁ is selected from thegroup consisting of hydrogen, and amino; and R₁₂ independently are eachoccurrence represents hydrogen, halo, or methyl.

In some embodiments, compounds of Formula II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are provided,wherein Y is selected from a group consisting of substituted cyclohexyl,cyclohexenyl, piperidinyl, and piperazinyl; R₁ is selected from thegroup consisting of hydrogen, —NH₂ halo, C₁₋₄ alkyl, C₃₋₄ cycloalkyl,and —CN; R₁₂ independently at each occurrence is selected from the groupconsisting of hydrogen, halo, C₁₋₄ alkyl, and amino; and R₅ is selectedfrom the group consisting of substituted or unsubstituted phenyl,cyclohexyl, cyclopentyl, thiazole, pyridyl, pyrimidyl and pyrazinyl,wherein the R₅ group may be substituted with up to three substituentsselected from halo, hydrogen, methyl, substituted aminocarbonyl andalkoxy.

In a representative embodiment, compounds of Formulas I or II, or astereoisomer, tautomer, or pharmaceutically acceptable salt thereof areprovided, selected from the group consisting ofN-(4-((1R,3R,4R,5S)-3-amino-4-hydroxy-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide.N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide,3-amino-N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide,andN-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide,and3-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide.

In other aspects, the present invention provides methods for treatingProvirus Integration of Maloney Kinase (PIM Kinase) related disorders ina human or animal subject in need of such treatment comprisingadministering to said subject an amount of a compound of Formula I or IIeffective to inhibit PIM activity in the subject.

In other aspects, the present invention provides methods for treatingPIM related disorders in a human or animal subject in need of suchtreatment comprising administering to said subject an amount of acompound of Formula I or II effective to reduce or prevent tumor growthin the subject.

In yet other aspects, the present invention provides methods fortreating PIM related disorders in a human or animal subject in need ofsuch treatment comprising administering to said subject an amount of acompound of Formula I or II effective to reduce or prevent tumor growthin the subject in combination with at least one additional agent for thetreatment of cancer.

In yet other aspects, the present invention provides therapeuticcompositions comprising at least one compound of Formula I or II incombination with one or more additional agents for the treatment ofcancer, as are commonly employed in cancer therapy.

The compounds of the invention are useful in the treatment of cancers,including hematopoietic malignancies, carcinomas (e.g., of the lungs,liver, pancreas, ovaries, thyroid, bladder or colon), melanoma, myeloiddisorders (e.g., myeloid leukemia, multiple myeloma anderythroleukemia), adenomas (e.g., villous colon adenoma), sarcomas(e.g., osteosarcoma), autoimmune diseases, allergic reactions and inorgan transplantation rejection syndromes.

The invention further provides compositions, methods of use, and methodsof manufacture as described in the detailed description of theinvention.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a graph showing the efficacy of the compound of Example 99from an evaluation in the KMS11-luc xenograft model, as described inExample 144.

FIG. 2 is a graph showing the efficacy of the compound of Example 70from an evaluation in the KMS11-luc xenograft model, as described inExample 144.

FIG. 3 is a graph showing the efficacy of the compound of Example 96from an evaluation in the KMS11-luc xenograft model, as described inExample 144.

DETAILED DESCRIPTION

In accordance with one aspect of the present invention, new compounds,and their stereoisomers, tautomers and pharmaceutically acceptablesalts, are provided of the Formula I:

-   -   wherein,    -   X₁, X₂, X₃ and X₄ are independently selected from CR₂ and N;        provided that at least one but not more than two of X₁, X₂, X₃        and X₄ are N;    -   Y is selected from a group consisting of cycloalkyl, partially        unsaturated cycloalkyl, and heterocycloalkyl, wherein each        member of said group may be substituted with up to four        substituents;    -   Z₂ and Z₃ are independently selected from CR₁₂ and N; provided        that not more than one of Z₂ and Z₃ can be N;    -   R₁ is selected from the group consisting of hydrogen, —NHR₃        halo, hydroxyl, alkyl, cyano, and nitro;    -   R₂ and R₁₂ independently at each occurrence are selected from        the group consisting of hydrogen, halo, hydroxyl, nitro, cyano,        SO₃H and substituted or unsubstituted alkyl, alkenyl, alkynyl,        alkoxy, amino, cycloalkyl, hetero cycloalkyl, and partially        saturated cycloalkyl;    -   R₃ is selected from the group consisting of hydrogen, —CO—R₄ and        substituted or unsubstituted alkyl, cycloalkyl, heterocyclyl,        aryl and heteroaryl;    -   R₄ is selected from the group consisting of alkyl, substituted        alkyl, alkoxy, substituted alkoxy, amino, substituted amino, and        alkylamino; and    -   R₅ represents a group selected from substituted or unsubstituted        aryl, C₃-C₇ cycloalkyl, heteroaryl, partially unsaturated        cycloalkyl and alkyl, wherein each said substituted R₅ group may        be substituted with up to four substituents selected from halo,        cyano, amino, C₁₋₄ alkyl, C₃₋₆ cycloalkyl, alkoxy, nitro,        carboxy, carbonyl, carboalkoxy, aminocarboxy, substituted        aminocarbonyl, aminosulfonyl, substituted aminosulfonyl and        alkoxyalkyl.

In some embodiments, new compounds of Formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein X₂ is N and X₁, X₃ and X₄ are CR₂.

In some embodiments, new compounds of Formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₂ is selected from hydrogen, methyl, ethyl, halo, cyano.

In some embodiments, compounds of formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein Z₂ and Z₃ are CR₁₂.

In some embodiments, compounds of formula I, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₁₂ is selected from hydrogen, halo, methyl, ethyl and cyano.

In other embodiments, new compounds, and their stereoisomers, tautomersand pharmaceutically acceptable salts, are provided of the Formula II

-   -   wherein,    -   Y is selected from a group consisting of cyclohexyl, partially        unsaturated cyclohexyl, and heterocyclo-C₅-alkyl, wherein each        member of said group may be substituted with up to four        substituents;    -   R₁ is selected from the group consisting of hydrogen, —NHR₃        halo, hydroxyl, alkyl, C₃₋₄ cycloalkyl, cyano, and nitro;    -   R₁₂ independently at each occurrence is selected from the group        consisting of hydrogen, halo, hydroxyl, amino, nitro, cyano,        SO₃H and substituted or unsubstituted alkyl, alkenyl, alkynyl,        alkoxy, amino, cycloalkyl, hetero cycloalkyl, and partially        saturated cycloalkyl;    -   R₃ is selected from the group consisting of hydrogen, —CO—R₄ and        substituted or unsubstituted alkyl, cycloalkyl, heterocyclyl,        aryl and heteroaryl;    -   R₄ is selected from the group consisting of alkyl, substituted        alkyl, alkoxy, substituted alkoxy, amino, substituted amino, and        alkylamino; and    -   R₅ is represents a group selected from hydrogen and substituted        or unsubstituted alkyl, C₆-cycloalkyl, aryl and heteroaryl.

In some embodiments, compounds of Formulas I or II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein Y is selected from a group consisting of substituted orunsubstituted cycloalkyl, cycloalkenyl, piperidinyl and piperazinyl,wherein each member of said group is substituted with up to foursubstituents. In some embodiments, compounds of Formulas I or II, or astereoisomer, tautomer, or pharmaceutically acceptable salt thereof areprovided wherein Y is selected from a group consisting of substituted orunsubstituted cyclohexyl, cyclohexynyl, and piperidinyl, wherein eachmember of said group is substituted with up to four substituents. Insome embodiments, Y is substituted with up to four substituents selectedfrom hydrogen, cyano, nitro, halo, hydroxyl, amino, alkoxy, substitutedamino, C₁₋₄ alkyl, C₁₋₄ halo alkyl and C₃₋₄ cycloalkyl. In yet otherembodiments, Y is substituted with up to four substituents selected frommethyl, propyl, i-propyl, ethyl, hydroxyl, amino, halo, monohalo C₁₋₃alkyl, trihalo C₁₋₃ alkyl and dihalo C₁₋₃ alkyl.

In some embodiments, compounds of Formulas I or II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₁ is hydrogen, amino or fluoro. In one embodiment are providedcompounds of Formula II selected from Table I or Table II.

In some embodiments, compounds of Formulas I or II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₅ is selected from substituted or unsubstituted aryl, C₅-C₆cycloalkyl, heteroaryl, partially unsaturated C₅-C₆ cycloalkyl and C₁-C₄alkyl, wherein each said group can be substituted with up to foursubstituents selected from halo, cyano, amino, C₁₋₄ alkyl, C₃₋₅cycloalkyl, alkoxy, nitro, carboxy, carbonyl, carboalkoxy, aminocarboxy,substituted aminocarbonyl, aminosulfonyl, substituted aminosulfonyl andalkoxyalkyl. In some embodiments, compounds of Formulas I or II, or astereoisomer, tautomer, or pharmaceutically acceptable salt thereof areprovided wherein R₅ is substituted or unsubstituted phenyl, wherein thephenyl group can be substituted with up to four substituents selectedfrom hydrogen, cyano, nitro, halo, hydroxyl, amino, alkoxy, substitutedamino, C₁₋₄ alkyl, C₁₋₄ halo alkyl and C₃₋₄ cycloalkyl. In someembodiments, compounds of Formulas I or II, or a stereoisomer, tautomer,or pharmaceutically acceptable salt thereof are provided wherein R₅ is2,6-difluororphenyl.

In some embodiments, compounds of Formulas I or II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein R₁₂ is selected from hydrogen, halo, methyl, ethyl and cyano. Insome embodiments, compounds of Formulas I or II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are providedwherein Y is selected from the group consisting of substituted orunsubstituted cyclohexyl, cyclohexenyl, piperidinyl, piperazinyl,wherein said the Y group may be substituted with up to threesubstituents selected from methyl, ethyl, hydroxyl, amino, and methoxy;R₁ is selected from the group consisting of hydrogen, and amino; and R₁₂independently are each occurrence represents hydrogen, halo, or methyl.

In some embodiments, compounds of Formula II, or a stereoisomer,tautomer, or pharmaceutically acceptable salt thereof are provided,wherein Y is selected from a group consisting of substituted cyclohexyl,cyclohexenyl, piperidinyl, and piperazinyl; R₁ is selected from thegroup consisting of hydrogen, —NH₂ halo, C₁₋₄ alkyl, C₃₋₄ cycloalkyl,and —CN; R₁₂ independently at each occurrence is selected from the groupconsisting of hydrogen, halo, C₁₋₄ alkyl, and amino; and R₅ is selectedfrom the group consisting of substituted or unsubstituted phenyl,cyclohexyl, cyclopentyl, thiazole, pyridyl, pyrimidyl and pyrazinyl,wherein the R₅ group may be substituted with up to three substituentsselected from halo, hydrogen, methyl, substituted aminocarbonyl andalkoxy.

A preferred embodiment of the present invention is a compound of Formula(II), wherein Y is cyclohexyl, substituted with one to threesubstitutents, said substituents preferably selected from hydroxyl,amino, C₁₋₄ alkyl or C₁₋₄ halo alkyl, and more preferably, selected frommethyl, hydroxyl, amino, and CF₃, and most preferably from methyl,amino, and hydroxy; R₁ is hydrogen, NH₂, or halo (preferably, R₁ ishydrogen, amino or fluoro, more preferably, R₁ is hydrogen); R₁₂ areeach independently hydrogen or halo (preferably, each R₁₂ is hydrogen,chloro or fluoro); R₅ is cyclohexyl, phenyl, or pyridyl, wherein saidcyclohexyl, said phenyl and said pyridyl are each independentlysubstituted with up to three substituents selected form halo, hydroxyl,C₁₋₄ alkyl, and C₁₋₄ alkoxy (preferably, R₅ is pyridyl or phenyl eachindependently substituted with up to three substitutents selected formhalo, hydroxyl, C₁₋₄ alkyl or C₁₋₄ alkoxy, more preferably, R₅ is phenylsubstituted with up to three substituents selected form halo, hydroxyl,C₁₋₄ alkoxy and C₁₋₄ alkyl, most preferably, phenyl substituted with upto three substitutents selected from fluoro, hydroxyl, methyl, ethyl,methoxy, or propoxy, most preferably, R₅ is 2,6-difluorophenyl.

Yet another preferred embodiment of the present invention provides acompound of Formula II, wherein Y is piperidinyl substituted withmethyl, hydroxyl, and amino; R₁ is hydrogen, NH₂, or fluoro; R₁₂independently at each occurrence is selected from the group consistingof hydrogen, and halo; and R₅ is pyridyl, fluoro pyridyl, cyclohexyl, orphenyl, wherein said phenyl is substituted with up to three substituentsselected from fluoro, hydroxyl, and methyl, preferably R₅ being difluorophenyl. In a further preferred embodiment preferably Y is3-amino-4-hydroxy-5-methylpiperidin-1-yl; R₁ is hydrogen; and R₅ is2,6-difluoro phenyl.

In a representative embodiment, preferred compounds of Formulas I or II,or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereofare selected from the group consisting ofN-(4-((3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((1R,3R,4S,5S)-3-amino-4-hydroxy-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamide;N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluoro-3-methylphenyl)-5-fluoropicolinamide;3-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((1R,3R,4R,5S)-3-amino-4-hydroxy-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide);N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((1R,3S)-3-aminocyclohexyl)-pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;and3-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide.

In other aspects, the present invention provides methods for treatingProvirus Integration of Maloney Kinase (PIM Kinase) related disorders ina human or animal subject in need of such treatment comprisingadministering to said subject an amount of a compound of Formula I or IIeffective to inhibit PIM activity in the subject. A preferred embodimentof the present invention provides a method for treating a condition bymodulation of Provirus Integration of Maloney Kinase (PIM Kinase)activity comprising administering to a patient in need of such treatmentan effective amount of a compound of Formula I.

In other aspects, the present invention provides methods for treatingPIM related disorders in a human or animal subject in need of suchtreatment comprising administering to said subject an amount of acompound of Formula I or II effective to reduce or prevent tumor growthin the subject. In yet other aspects, the present invention providesmethods for treating PIM related disorders in a human or animal subjectin need of such treatment comprising administering to said subject anamount of a compound of Formula I or II effective to reduce or preventtumor growth in the subject in combination with at least one additionalagent for the treatment of cancer.

In yet other aspect, the present invention provides therapeuticcompositions comprising at least one compound of Formula I or II incombination with one or more additional agents for the treatment ofcancer, as are commonly employed in cancer therapy. The presentinvention thus provides a pharmaceutical composition comprising acompound of Formula I or Formula II. A preferred embodiment of thisaspect provides a pharmaceutical composition comprising a compoundselected fromN-(4-((3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((1R,3R,4S,5S)-3-amino-4-hydroxy-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamide;N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((1R,3R,4R,5S)-3-amino-4-hydroxy-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide);N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((1R,3S)-3-aminocyclohexyl)-pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;3-amino-N-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide;andN-(4-((3R,4R,5S)-3-amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluoro-3-methylphenyl)-5-fluoropicolinamide.Another preferred embodiment provides a pharmaceutical compositionfurther comprising an additional agent for the treatment of cancer,wherein preferably the additional agent is selected from irinotecan,topotecan, gemcitabine, 5-fluorouracil, leucovorin carboplatin,cisplatin, taxanes, tezacitabine, cyclophosphamide, vinca alkaloids,imatinib (Gleevec), anthracyclines, rituximab, and trastuzumab.

The compounds of the invention are useful in the treatment of cancers,including hematopoietic malignancies, carcinomas (e.g., of the lungs,liver, pancreas, ovaries, thyroid, bladder or colon), melanoma, myeloiddisorders (e.g., myeloid leukemia, multiple myeloma anderythroleukemia), adenomas (e.g., villous colon adenoma), sarcomas(e.g., osteosarcoma), autoimmune diseases, allergic reactions and inorgan transplantation rejection syndromes.

In yet another aspect of the present invention is provided a use of acompound of Formula I or Formula II for preparing a medicament fortreating a condition by modulation of Provirus Integration of MaloneyKinase (PIM Kinase) activity. In a preferred embodiment of this aspectof the invention the condition is a cancer selected from carcinoma ofthe lungs, pancreas, thyroid, ovarian, bladder, breast, prostate, orcolon, melanoma, myeloid leukemia, multiple myeloma and erythroleukemia, villous colon adenoma, and osteosarcoma.

In another aspect, the present invention relates to methods ofinhibiting the activity of at least one kinase selected from the groupconsisting of Pim1, Pim2 and Pim3, in a subject, or treating abiological condition mediated by at least one of Pim1, Pim2 and Pim3, ina human or animal subject in need of such treatment, comprisingadministering to the subject at least one compound of Formula I or II inan amount effective to inhibit the kinase in the subject. Thetherapeutic compounds are useful for treating patients with a need forsuch inhibitors (e.g., those suffering from cancer mediated by abnormalserine/threonine kinase receptor signaling).

Definitions

“PIM inhibitor” is used herein to refer to a compound that exhibits anIC₅₀ with respect to PIM Kinase activity of no more than about 100 μMand more typically not more than about 50 μM, as measured in the PIMdepletion assays described hereinbelow.

The phrase “alkyl” refers to alkyl groups that do not containheteroatoms. Thus the phrase includes straight chain alkyl groups suchas methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl and the like. The phrase also includes branchedchain isomers of straight chain alkyl groups, including but not limitedto, the following which are provided by way of example: —CH(CH₃)₂,—CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃, —C(CH₂CH₃)₃, —CH₂CH(CH₃)₂,—CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂, —CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃,—CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂, —CH₂CH₂CH(CH₃)(CH₂CH₃),—CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃, —CH₂CH₂C(CH₂CH₃)₃,—CH(CH₃)CH₂—CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)₂,—CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. Thus the phrase alkylgroups includes primary alkyl groups, secondary alkyl groups, andtertiary alkyl groups. Preferred alkyl groups include straight andbranched chain alkyl groups having 1 to 12 carbon atoms. A preferred“alkyl” definition refers to C₁₋₄ straight chain alkyl groups such asmethyl, ethyl, n-propyl, and n-butyl. The preferred alkyl definitionalso includes C₃₋₅ branched alkyl groups, including CH(CH₃)₂,CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, C(CH₃)₃, CH(CH₃)CH₂CH₂CH₃, CH(CH₃)CH(CH₃)₂,CH₂CH(CH₃)CH₂CH₃, CH₂CH₂CH(CH₃)₂, and CH(CH₂CH₃)₂, etc.

The term “alkenyl” refers to alkyl groups as defined above, whereinthere is at least one point of unsaturation, i.e., wherein two adjacentcarbon atoms are attached by a double bond. The term “alkynyl” refers toalkyl groups wherein two adjacent carbon atoms are attached by a triplebond. The term “alkoxy” refers to —OR, wherein R is alkyl.

As used herein, the term “halogen” or “halo” refers to chloro, bromo,fluoro and iodo groups. “Haloalkyl” refers to an alkyl radicalsubstituted with one or more halogen atoms. The term “haloalkyl” thusincludes monohalo alkyl, dihalo alkyl, trihalo alkyl and the like.Representative monohalo alkyl groups include —CH₂F, —CH₂Cl, —CH₂CH₂F,—CH₂CH₂Cl, —CH(F)CH₃, —CH(Cl)CH₃; representative dihalo alkyl groupsinclude CHCl₂, —CHF₂, —CCl₂CH₃, —CH(Cl)CH₂Cl, —CH₂CHCl₂, —CH₂CHF₂;representative trihalo alkyl groups include —CCl₃, —CF₃, —CCl₂CH₂Cl,—CF₂CH₂F, —CH(Cl)CHCl₂, —CH(F)CHF₂; and representative perhalo alkylgroups include —CCl₃, —CF₃, —CCl₂CCl₃, —CF₂CF₃.

“Amino” refers herein to the group —NH₂. The term “alkylamino” refersherein to the group —NRR′ where R and R′ are each independently selectedfrom hydrogen or a lower alkyl. The term “arylamino” refers herein tothe group —NRR′ where R is aryl and R′ is hydrogen, a lower alkyl, or anaryl. The term “aralkylamino” refers herein to the group —NRR′ where Ris a lower aralkyl and R′ is hydrogen, a loweralkyl, an aryl, or aloweraralkyl. The term cyano refers to the group —CN. The term nitrorefers to the group —NO₂.

The term “alkoxyalkyl” refers to the group -alk₁-O-alk₂ where alk₁ isalkyl or alkenyl, and alk₂ is alkyl or alkenyl. The term“loweralkoxyalkyl” refers to an alkoxyalkyl where alk₁ is loweralkyl orloweralkenyl, and alk₂ is loweralkyl or loweralkenyl. The term“aryloxyalkyl” refers to the group -alkyl-O-aryl. The term“aralkoxyalkyl” refers to the group -alkylenyl-O-aralkyl, where aralkylis a loweraralkyl.

The term “aminocarbonyl” refers herein to the group —C(O)—NH₂.“Substituted aminocarbonyl” refers herein to the group —C(O)—NRR′ whereR is loweralkyl and R′ is hydrogen or a loweralkyl. In some embodiments,R and R′, together with the N atom attached to them may be takentogether to form a “heterocycloalkylcarbonyl” group. The term“arylaminocarbonyl” refers herein to the group —C(O)—NRR′ where R is anaryl and R′ is hydrogen, loweralkyl or aryl. “aralkylaminocarbonyl”refers herein to the group —C(O)—NRR′ where R is loweraralkyl and R′ ishydrogen, loweralkyl, aryl, or loweraralkyl.

“Aminosulfonyl” refers herein to the group —S(O)₂—NH₂. “Substitutedaminosulfonyl” refers herein to the group —S(O)₂—NRR′ where R isloweralkyl and R′ is hydrogen or a loweralkyl. The term“aralkylaminosulfonlyaryl” refers herein to the group-aryl-S(O)₂—NH-aralkyl, where the aralkyl is loweraralkyl.

“Carbonyl” refers to the divalent group —C(O)—. “Carboxy” refers to—C(═O)—OH. “Alkoxycarbonyl” refers to ester —C(═O)—OR wherein R isalkyl. “Loweralkoxycarbonyl” refers to ester —C(═O)—OR wherein R isloweralkyl. “Cycloalkyloxycarbonyl” refers to —C(═O)—OR wherein R iscycloalkyl.

“Cycloalkyl” refers to a mono- or polycyclic, carbocyclic alkylsubstituent. Carbocycloalkyl groups are cycloalkyl groups in which allring atoms are carbon. Typical cycloalkyl substituents have from 3 to 8backbone (i.e., ring) atoms in which each backbone atom is either carbonor a heteroatom. The term “heterocycloalkyl” refers herein to cycloalkylsubstituents that have from 1 to 5, and more typically from 1 to 4heteroatoms in the ring structure. Suitable heteroatoms employed incompounds of the present invention are nitrogen, oxygen, and sulfur.Representative heterocycloalkyl moieties include, for example,morpholino, piperazinyl, piperidinyl and the like. Carbocycloalkylgroups are cycloalkyl groups in which all ring atoms are carbon. Whenused in connection with cycloalkyl substituents, the term “polycyclic”refers herein to fused and non-fused alkyl cyclic structures. The term“partially unsaturated cycloalkyl”, “partially saturated cycloalkyl”,and “cycloalkenyl” all refer to a cycloalkyl group wherein there is atleast one point of unsaturation, i.e., wherein to adjacent ring atomsare connected by a double bond or a triple bond. Illustrative examplesinclude cyclohexynyl, cyclohexynyl, cyclopropenyl, cyclobutynyl, and thelike.

The terms “substituted heterocycle”, “heterocyclic group” or“heterocycle” as used herein refers to any 3- or 4-membered ringcontaining a heteroatom selected from nitrogen, oxygen, and sulfur or a5- or 6-membered ring containing from one to three heteroatoms selectedfrom the group consisting of nitrogen, oxygen, or sulfur; wherein the5-membered ring has 0-2 double bonds and the 6-membered ring has 0-3double bonds; wherein the nitrogen and sulfur atom maybe optionallyoxidized; wherein the nitrogen and sulfur heteroatoms may be optionallyquarternized; and including any bicyclic group in which any of the aboveheterocyclic rings is fused to a benzene ring or another 5- or6-membered heterocyclic ring independently defined above. The term or“heterocycloalkyl” as used herein refers to a 5- or 6-membered ringcontaining from one to three heteroatoms selected from the groupconsisting of nitrogen, oxygen, or sulfur, wherein the ring has nodouble bonds. For example, the term heterocyclo-C₅-alkyl refers to a6-membered ring containing 5 carbon atoms and a heteroatom, such as N.The term “heterocycle” thus includes rings in which nitrogen is theheteroatom as well as partially and fully-saturated rings. Preferredheterocycles include, for example: diazapinyl, pyrryl, pyrrolinyl,pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazoyl,imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, pyrazinyl,piperazinyl, N-methyl piperazinyl, azetidinyl, N-methylazetidinyl,pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl,isoazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl,benzothiazolyl, benzoxazolyl, furyl, thienyl, triazolyl andbenzothienyl.

Heterocyclic moieties can be unsubstituted or monosubstituted ordisubstituted or trisubstituted with various substituents independentlyselected from hydroxy, halo, oxo (C═O), alkylimino (RN═, wherein R is aloweralkyl or loweralkoxy group), amino, alkylamino, dialkylamino,acylaminoalkyl, alkoxy, thioalkoxy, polyalkoxy, loweralkyl, cycloalkylor haloalkyl.

The heterocyclic groups may be attached at various positions as will beapparent to those having skill in the organic and medicinal chemistryarts in conjunction with the disclosure herein.

Representative heterocyclics include, for example, imidazolyl, pyridyl,piperazinyl, piperidinyl, azetidinyl, thiazolyl, furanyl, triazolylbenzimidazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl,quinazolinyl, quinoxalinyl, phthalazinyl, indolyl, naphthpyridinyl,indazolyl, and quinolizinyl.

“Aryl” refers to optionally substituted monocyclic and polycyclicaromatic groups having from 3 to 14 backbone carbon or hetero atoms, andincludes both carbocyclic aryl groups and heterocyclic aryl groups.Carbocyclic aryl groups are aryl groups in which all ring atoms in thearomatic ring are carbon. The term “heteroaryl” refers herein to arylgroups having from 1 to 4 heteroatoms as ring atoms in an aromatic ringwith the remainder of the ring atoms being carbon atoms. When used inconnection with aryl substituents, the term “polycyclic aryl” refersherein to fused and non-fused cyclic structures in which at least onecyclic structure is aromatic, such as, for example, benzodioxozolo(which has a heterocyclic structure fused to a phenyl group, i.e., andthe like. Exemplary aryl moieties employed as substituents in compoundsof the present invention include phenyl, pyridyl, pyrimidinyl,thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl,triazolyl, thiophenyl, furanyl, quinolinyl, purinyl, naphthyl,benzothiazolyl, benzopyridyl, and benzimidazolyl, and the like.

“Optionally substituted” or “substituted” refers to the replacement ofone or more hydrogen atoms with a monovalent or divalent radical.Suitable substitution groups include, for example, hydroxy, nitro,amino, imino, cyano, halo, thio, sulfonyl, thioamido, amidino, imidino,oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido,carboxyl, formyl, loweralkyl, haloloweralkyl, loweralkylamino,haloloweralkylamino, loweralkoxy, haloloweralkoxy, loweralkoxyalkyl,alkylcarbonyl, aminocarbonyl, arylcarbonyl, aralkylcarbonyl,heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio, aminoalkyl,cyanoalkyl, aryl and the like.

The substitution group can itself be substituted. The group substitutedonto the substitution group can be carboxyl, halo; nitro, amino, cyano,hydroxy, loweralkyl, loweralkoxy, aminocarbonyl, —SR, thioamido, —SO₃H,—SO₂R or cycloalkyl, where R is typically hydrogen, hydroxyl orloweralkyl.

When the substituted substituent includes a straight chain group, thesubstitution can occur either within the chain (e.g., 2-hydroxypropyl,2-aminobutyl, and the like) or at the chain terminus (e.g.,2-hydroxyethyl, 3-cyanopropyl, and the like). Substituted substituentscan be straight chain, branched or cyclic arrangements of covalentlybonded carbon or heteroatoms. It is understood that the abovedefinitions are not intended to include impermissible substitutionpatterns (e.g., methyl substituted with five fluoro groups or a halogenatom substituted with another halogen atom). Such impermissiblesubstitution patterns are well known to the skilled artisan.

It will also be apparent to those skilled in the art that the compoundsof the invention, or their stereoisomers, as well as thepharmaceutically acceptable salts, esters, metabolites and prodrugs ofany of them, may be subject to tautomerization and may therefore existin various tautomeric forms wherein a proton of one atom of a moleculeshifts to another atom and the chemical bonds between the atoms of themolecules are consequently rearranged. See, e.g., March, AdvancedOrganic Chemistry: Reactions, Mechanisms and Structures, Fourth Edition,John Wiley & Sons, pages 69-74 (1992). As used herein, the term“tautomer” refers to the compounds produced by the proton shift, and itshould be understood that the all tautomeric forms, insofar as they mayexist, are included within the invention.

The compounds of the invention, or their tautomers, as well as thepharmaceutically acceptable salts, esters, metabolites and prodrugs ofany of them, may comprise asymmetrically substituted carbon atoms. Suchasymmetrically substituted carbon atoms can result in the compounds ofthe invention existing in enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, such as in (R)- or (S)-forms. As a result, all suchpossible isomers, individual stereoisomers in their optically pureforms, mixtures thereof, racemic mixtures (or “racemates”), mixtures ofdiastereomers, as well as single diastereomers of the compounds of theinvention are included in the present invention. The terms “S” and “R”configuration, as used herein, are as defined by the IUPAC 1974RECOMMENDATIONS FOR SECTION E, FUNDAMENTAL STEREOCHEMISTRY, Pure Appl.Chem. 45:13-30 (1976). The terms α and β are employed for ring positionsof cyclic compounds. The α-side of the reference plane is that side onwhich the preferred substituent lies at the lower numbered position.Those substituents lying on the opposite side of the reference plane areassigned β descriptor. It should be noted that this usage differs fromthat for cyclic stereoparents, in which “α” means “below the plane” anddenotes absolute configuration. The terms α and β configuration, as usedherein, are as defined by the CHEMICAL ABSTRACTS INDEX GUIDE-APPENDIX IV(1987) paragraph 203.

As used herein, the term “pharmaceutically acceptable salts” refers tothe nontoxic acid or alkaline earth metal salts of the compounds ofFormula I or II. These salts can be prepared in situ during the finalisolation and purification of the compounds of Formula I or II, or byseparately reacting the base or acid functions with a suitable organicor inorganic acid or base, respectively. Representative salts includebut are not limited to the following: acetate, adipate, alginate,citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,camphorate, camphorsulfonate, digluconate, cyclopentanepropionate,dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylproionate, picrate, pivalate, propionate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate andundecanoate. Also, the basic nitrogen-containing groups can bequaternized with such agents as loweralkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides, and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethylbromides, and others. Water or oil-soluble or dispersible products arethereby obtained.

Examples of acids which may be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulfuric acid and phosphoric acid and such organicacids as oxalic acid, maleic acid, methanesulfonic acid, succinic acidand citric acid. Basic addition salts can be prepared in situ during thefinal isolation and purification of the compounds of formula (I), orseparately by reacting carboxylic acid moieties with a suitable basesuch as the hydroxide, carbonate or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia, or an organic primary,secondary or tertiary amine. Pharmaceutically acceptable salts include,but are not limited to, cations based on the alkali and alkaline earthmetals, such as sodium, lithium, potassium, calcium, magnesium, aluminumsalts and the like, as well as nontoxic ammonium, quaternary ammonium,and amine cations, including, but not limited to ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, ethylamine, and the like. Otherrepresentative organic amines useful for the formation of base additionsalts include diethylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine and the like.

As used herein, the term “pharmaceutically acceptable ester” refers toesters, which hydrolyze in vivo and include those that break downreadily in the human body to leave the parent compound or a saltthereof. Suitable ester groups include, for example, those derived frompharmaceutically acceptable aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.Examples of particular esters include formates, acetates, propionates,butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention. The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compound of the above formula,for example by hydrolysis in blood. A thorough discussion is provided inT. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14of the A.C.S. Symposium Series, and in Edward B. Roche, ed.,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are incorporatedherein by reference

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F ³¹F, ³²F, ³⁵S, ³⁶Cl, ¹²⁵Irespectively. The invention includes various isotopically labeledcompounds as defined herein, for example those into which radioactiveisotopes, such as ³H, ¹³C, and ¹⁴C, are present. Such isotopicallylabelled compounds are useful in metabolic studies (with ¹⁴C), reactionkinetic studies (with, for example ²H or ³H), detection or imagingtechniques, such as positron emission tomography (PET) or single-photonemission computed tomography (SPECT) including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Inparticular, an ¹⁸F or labeled compound may be particularly desirable forPET or SPECT studies. Isotopically labeled compounds of this inventionand prodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent.

Further, substitution with heavier isotopes, particularly deuterium(i.e., ²H or D) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements or an improvement in therapeutic index. Itis understood that deuterium in this context is regarded as asubstituent of a compound of the formula (I). The concentration of sucha heavier isotope, specifically deuterium, may be defined by theisotopic enrichment factor. The term “isotopic enrichment factor” asused herein means the ratio between the isotopic abundance and thenatural abundance of a specified isotope. If a substituent in a compoundof this invention is denoted deuterium, such compound has an isotopicenrichment factor for each designated deuterium atom of at least 3500(52.5% deuterium incorporation at each designated deuterium atom), atleast 4000 (60% deuterium incorporation), at least 4500 (67.5% deuteriumincorporation), at least 5000 (75% deuterium incorporation), at least5500 (82.5% deuterium incorporation), at least 6000 (90% deuteriumincorporation), at least 6333.3 (95% deuterium incorporation), at least6466.7 (97% deuterium incorporation), at least 6600 (99% deuteriumincorporation), or at least 6633.3 (99.5% deuterium incorporation).

Isotopically-labeled compounds of formula (I) can generally be preparedby conventional techniques known to those skilled in the art or byprocesses analogous to those described in the accompanying Examples andPreparations using an appropriate isotopically-labeled reagents in placeof the non-labeled reagent previously employed.

It will be apparent to those skilled in the art that the compounds ofthe invention, or their tautomers, prodrugs and stereoisomers, as wellas the pharmaceutically acceptable salts, esters and prodrugs of any ofthem, may be processed in vivo through metabolism in a human or animalbody or cell to produce metabolites. The term “metabolite” as usedherein refers to the formula of any derivative produced in a subjectafter administration of a parent compound. The derivatives may beproduced from the parent compound by various biochemical transformationsin the subject such as, for example, oxidation, reduction, hydrolysis,or conjugation and include, for example, oxides and demethylatedderivatives. The metabolites of a compound of the invention may beidentified using routine techniques known in the art. See, e.g.,Bertolini, G. et al., J. Med. Chem. 40:2011-2016 (1997); Shan, D. etal., J. Pharm. Sci. 86(7):765-767; Bagshawe K., Drug Dev. Res.34:220-230 (1995); Bodor, N., Advances in Drug Res. 13:224-331 (1984);Bundgaard, H., Design of Prodrugs (Elsevier Press 1985); and Larsen, I.K., Design and application of Prodrugs, Drug Design and Development(Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991). Itshould be understood that individual chemical compounds that aremetabolites of the compounds of formula I, formula II, or theirtautomers, prodrugs and stereoisomers, as well as the pharmaceuticallyacceptable salts, esters and prodrugs of any of them, are includedwithin the invention.

The term “cancer” refers to cancer diseases that can be beneficiallytreated by the inhibition of Pim kinase, including, for example, solidcancers, such as carcinomas (e.g., of the lungs, pancreas, thyroid,ovarian, bladder, breast, prostate, or colon), melanomas, myeloiddisorders (e.g., myeloid leukemia, multiple myeloma anderythroleukemia), adenomas (e.g., villous colon adenoma) and sarcomas(e.g., osteosarcoma).

Synthetic Methods

The compounds of the invention can be obtained through procedures knownto the skilled in the art. For example, as shown in Scheme 1,cyclohexanediones can be converted via monotriflates to thecorresponding cyclohexenoneboronate esters which can undergo palladiummediated carbon bond formation with 4-chloro, 3-nitro pyridine to yieldnitropyridine substituted cyclohexenones I. Reduction of the enonefunctionality can yield a cyclohexenol II which upon alcohol protection,nitro and alkene reduction, amide coupling and deprotection can yieldcyclohexanol amides III. Cyclohexenol II can also undergo Mitsunobureaction with phthalimide to yield a protected aminocyclohexene IV.Following nitro and alkene reduction, phthalimide protectedaminocyclohexyl pyridyl aniline Va can undergo amide coupling anddeprotection, to yield aminocyclohexane amides VI. The corresponding Bocprotected aminocyclohexane pyridyl aniline Vb can also be prepared fromcyclohexenol II in the following manner: alcohol protection, alkene andnitro reduction, pyridyl amine Cbz protection, silyl ether deprotection,Dess-Martin oxidation to the cyclohexanone, reductive amination withbenzylamine, Cbz and Bn deprotection and primary aliphatic amine Bocprotection. In the amide products III and VI, if R₂ is halo or triflate,the amides III and VI can be further modified by standard modificationsto introduce substituted aryls, alkyls and heteroaryls at R₂. Forexample, if R₂ is Br, by reaction with boronic acids or organometallicreagents, or conversion to the corresponding boronate ester and reactionwith aryl/heteroaryl halides or triflates, a variety of R₂ modificationsare possible.

Alternatively, as shown in Scheme 2, cyclohexenol II can be dehydratedyielding a cyclohexadiene which upon epoxidation (via bromohydrinformation and HBr elimination or from mCPBA directly) and azide epoxideopening yields cyclohexenyl azido alcohol VI. Cyclohexenyl azido alcoholVI can be converted to the trans protected amino hydroxy aniline VIIa byazide reduction, alcohol protection and alkene and nitro reduction.Alternatively, the cyclohexenyl azido alcohol VI can be converted to theprotected cis amino hydroxy aniline VIIb by azide reduction and Bocprotection, alcohol mesylation and intramolecular cyclization to the ciscyclic carbamate, followed by Boc protection and alkene and nitroreduction. The resulting cyclohexylpyridyl anilines VIIa and VIIb can beconverted to the corresponding pyridine amides VIIIa and VIIIb by amidecoupling, acetate or cyclic carbamate cleavage and Boc deprotection. IfR₂ is halo or triflate, the amides VIIIa and VIIIb can be furthermodified by standard modifications to introduce substituted aryls,alkyls and heteroaryls at R₂ after amide bond formation and prior tofull deprotection. For example, if R₂ is Br, by reaction with boronicacids or organometallic reagents, or conversion to the correspondingboronate ester and reaction with aryl/heteroaryl halides or triflates, avariety of R₂ modifications are possible.

Alternatively, as shown in Scheme 3, trisubstituted 5-alkyl, 4-hydroxy,3-aminopiperidines can be prepared and modified to yield trisubstituted5-alkyl, 4-hydroxy, 3-aminopiperidinyl pyridine amides IX as follows.Reaction of Garner's aldehyde with(R)-4-benzyl-3-propionyloxazolidin-2-one followed by TBS protection ofthe resulting alcohol affords compound X. Reduction of the oxazolidinonefollowed by introduction of the azide group yields intermediate XI.Deprotection under acidic conditions reveals the corresponding aminoalcohol, which upon protection with the Boc group followed by mesylationof the primary alcohol yields intermediate XII. Reduction of the azideaffords formation of the piperidine which is subsequently reacted with4-chloro-3-nitropyridine, reduced to the amine, coupled with thecorresponding carboxylic acid and deprotected to provide trisubstituted5-methyl,4-hydroxy-3-aminopiperidinyl pyridine amides IX. If R₁ is haloor triflate, the amide IX can be further modified by standardmodifications to introduce substituted aryls, alkyls and heteroaryls atR₁ after amide bond formation and prior to full deprotection. Forexample, if R₁ is Br, by reaction with boronic acids or organometallicreagents, or conversion to the corresponding boronate ester and reactionwith aryl/heteroaryl halides or triflates, a variety of R₁ modificationsare possible.

Alternatively, as shown in Scheme 4, trisubstituted 5-methyl, 4-hydroxy,3-aminopiperidines can also be prepared and modified to yieldtrisubstituted 5-methyl, 4-hydroxy, 3-aminopiperidinyl amides XIII asfollows. Reaction of crotyl boronate esters with SerOBn aldehydefollowed by cyclic carbamate formation, alkene oxidative cleavage andreduction yields hydroxyl compound XIV. Benzyl deprotection followed bybistosylation and reaction with p-methoxybenzylamine, and aminedeprotection yields piperidine XV. Reaction of substituted piperidine XVwith halo nitro substituted arenes or heteroarenes followed by carbamateprotection, nitro reduction, amide coupling, cyclic carbamate openingand deprotection yields trisubstituted 5-methyl, 4-hydroxy,3-aminopiperidinyl amides XIII. If R₃ is halo or triflate, the amide XVcan be further modified by standard modifications to introducesubstituted aryls, alkyls and heteroaryls at R₃. For example, if R₃ isBr, by reaction with boronic acids or organometallic reagents, orconversion to the corresponding boronate ester and reaction witharyl/heteroaryl halides or triflates, a variety of R₃ modifications arepossible.

The compounds of the invention are useful in vitro and/or in vivo ininhibiting the growth of cancer cells. The compounds may be used aloneor in compositions together with a pharmaceutically acceptable carrieror excipient. Suitable pharmaceutically acceptable carriers orexcipients include, for example, processing agents and drug deliverymodifiers and enhancers, such as, for example, calcium phosphate,magnesium stearate, talc, monosaccharides, disaccharides, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidinone, lowmelting waxes, ion exchange resins, and the like, as well ascombinations of any two or more thereof. Other suitable pharmaceuticallyacceptable excipients are described in “Remington's PharmaceuticalSciences,” Mack Pub. Co., New Jersey (1991), incorporated herein byreference.

Effective amounts of the compounds of the invention generally includeany amount sufficient to detectably inhibit Pim activity by any of theassays described herein, by other Pim kinase activity assays known tothose having ordinary skill in the art or by detecting an inhibition oralleviation of symptoms of cancer. The amount of active ingredient thatmay be combined with the carrier materials to produce a single dosageform will vary depending upon the host treated and the particular modeof administration. It will be understood, however, that the specificdose level for any particular patient will depend upon a variety offactors including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,route of administration, rate of excretion, drug combination, and theseverity of the particular disease undergoing therapy. Thetherapeutically effective amount for a given situation can be readilydetermined by routine experimentation and is within the skill andjudgment of the ordinary clinician.

For purposes of the present invention, a therapeutically effective dosewill generally be a total daily dose administered to a host in single ordivided doses may be in amounts, for example, of from 0.001 to 1000mg/kg body weight daily and more preferred from 1.0 to 30 mg/kg bodyweight daily. Dosage unit compositions may contain such amounts ofsubmultiples thereof to make up the daily dose.

The compounds of the present invention may be administered orally,parenterally, sublingually, by aerosolization or inhalation spray,rectally, or topically in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired. Topical administration may also involve the useof transdermal administration such as transdermal patches orionophoresis devices. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-propanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols, which are solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose lactose or starch. Such dosage forms may also comprise, as isnormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavoring, and perfuming agents.

The compounds of the present invention can also be administered in theform of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any nontoxic, physiologically acceptableand metabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to acompound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.W., p.33 et seq. (1976).

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other agents used in the treatment of cancer. The compoundsof the present invention are also useful in combination with knowntherapeutic agents and anti-cancer agents, and combinations of thepresently disclosed compounds with other anti-cancer or chemotherapeuticagents are within the scope of the invention. Examples of such agentscan be found in Cancer Principles and Practice of Oncology, V. T. Devitaand S. Hellman (editors), 6^(th) edition (Feb. 15, 2001), LippincottWilliams & Wilkins Publishers. A person of ordinary skill in the artwould be able to discern which combinations of agents would be usefulbased on the particular characteristics of the drugs and the cancerinvolved. Such anti-cancer agents include, but are not limited to, thefollowing: estrogen receptor modulators, androgen receptor modulators,retinoid receptor modulators, cytotoxic/cytostatic agents,antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoAreductase inhibitors and other angiogenesis inhibitors, inhibitors ofcell proliferation and survival signaling, apoptosis inducing agents andagents that interfere with cell cycle checkpoints. The compounds of theinvention are also useful when co-administered with radiation therapy.

Therefore, in one embodiment of the invention, the compounds of theinvention are also used in combination with known anticancer agentsincluding, for example, estrogen receptor modulators, androgen receptormodulators, retinoid receptor modulators, cytotoxic agents,antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoAreductase inhibitors, HIV protease inhibitors, reverse transcriptaseinhibitors, and other angiogenesis inhibitors.

In certain presently preferred embodiments of the invention,representative agents useful in combination with the compounds of theinvention for the treatment of cancer include, for example, irinotecan,topotecan, gemcitabine, 5-fluorouracil, leucovorin carboplatin,cisplatin, taxanes, tezacitabine, cyclophosphamide, vinca alkaloids,imatinib (Gleevec), anthracyclines, rituximab, trastuzumab, as well asother cancer chemotherapeutic agents.

The above compounds to be employed in combination with the compounds ofthe invention will be used in therapeutic amounts as indicated in thePhysicians' Desk Reference (PDR) 47th Edition (1993), which isincorporated herein by reference, or such therapeutically useful amountsas would be known to one of ordinary skill in the art.

The compounds of the invention and the other anticancer agents can beadministered at the recommended maximum clinical dosage or at lowerdoses. Dosage levels of the active compounds in the compositions of theinvention may be varied so as to obtain a desired therapeutic responsedepending on the route of administration, severity of the disease andthe response of the patient. The combination can be administered asseparate compositions or as a single dosage form containing both agents.When administered as a combination, the therapeutic agents can beformulated as separate compositions, which are given at the same time ordifferent times, or the therapeutic agents, can be given as a singlecomposition.

In one embodiment, the invention provides a method of inhibiting Pim1,Pim2 or Pim3 in a human or animal subject. The method includesadministering an effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, of any of the embodiments of compounds ofFormula I or II to a subject in need thereof.

The present invention will be understood more readily by reference tothe following examples, which are provided by way of illustration andare not intended to be limiting of the present invention.

EXAMPLES

Referring to the examples that follow, compounds of the preferredembodiments were synthesized using the methods described herein, orother methods, which are known in the art.

The compounds and/or intermediates were characterized by highperformance liquid chromatography (HPLC) using a Waters Milleniumchromatography system with a 2695 Separation Module (Milford, Mass.).The analytical columns were reversed phase Phenomenex Luna C18-5μ,4.6×50 mm, from Alltech (Deerfield, Ill.). A gradient elution was used(flow 2.5 mL/min), typically starting with 5% acetonitrile/95% water andprogressing to 100% acetonitrile over a period of 10 minutes. Allsolvents contained 0.1% trifluoroacetic acid (TFA). Compounds weredetected by ultraviolet light (UV) absorption at either 220 or 254 nm.HPLC solvents were from Burdick and Jackson (Muskegan, Mich.), or FisherScientific (Pittsburgh, Pa.).

In some instances, purity was assessed by thin layer chromatography(TLC) using glass or plastic backed silica gel plates, such as, forexample, Baker-Flex Silica Gel 1B2-F flexible sheets. TLC results werereadily detected visually under ultraviolet light, or by employingwell-known iodine vapor and other various staining techniques.

Mass spectrometric analysis was performed on one of three LCMSinstruments: a Waters System (Alliance HT HPLC and a Micromass ZQ massspectrometer; Column. Eclipse XDB-C18, 2.1×50 mm; gradient: 5-95% (or35-95%, or 65-95% or 95-95%) acetonitrile in water with 0.05% TFA over a4 min period; flow rate 0.8 mL/min; molecular weight range 200-1500;cone Voltage 20 V; column temperature 40° C.), another Waters System(ACQUITY UPLC system and a ZQ 2000 system; Column. ACQUITY UPLC HSS-C18,1.8 um, 2.1×50 mm; gradient: 5-95% (or 35-95%, or 65-95% or 95-95%)acetonitrile in water with 0.05% TFA over a 1.3 min period; flow rate1.2 mL/min; molecular weight range 150-850; cone Voltage 20 V; columntemperature 50° C.) or a Hewlett Packard System (Series 1100 HPLC;Column: Eclipse XDB-C18, 2.1×50 mm; gradient: 5-95% acetonitrile inwater with 0.05% TFA over a 4 min period; flow rate 0.8 mL/min;molecular weight range 150-850; cone Voltage 50 V; column temperature30° C.). All masses were reported as those of the protonated parentions.

Nuclear magnetic resonance (NMR) analysis was performed on some of thecompounds with a Varian 400 MHz NMR (Palo Alto, Calif.). The spectralreference was either TMS or the known chemical shift of the solvent.

Preparative separations are carried out using a Flash 40 chromatographysystem and KP-Sil, 60A (Biotage, Charlottesville, Va.), or by flashcolumn chromatography using silica gel (230-400 mesh) packing material,or by HPLC using a Waters 2767 Sample Manager, C-18 reversed phasecolumn, 30×50 mm, flow 75 mL/min. Typical solvents employed for theFlash 40 Biotage system and flash column chromatography aredichloromethane, methanol, ethyl acetate, hexane, acetone, aqueousammonia (or ammonium hydroxide), and triethyl amine. Typical solventsemployed for the reverse phase HPLC are varying concentrations ofacetonitrile and water with 0.1% trifluoroacetic acid.

It should be understood that the organic compounds according to thepreferred embodiments may exhibit the phenomenon of tautomerism. As thechemical structures within this specification can only represent one ofthe possible tautomeric forms, it should be understood that thepreferred embodiments encompasses any tautomeric form of the drawnstructure.

It is understood that the invention is not limited to the embodimentsset forth herein for illustration, but embraces all such forms thereofas come within the scope of the above disclosure.

The examples below as well as throughout the application, the followingabbreviations have the following meanings. If not defined, the termshave their generally accepted meanings.

ABBREVIATIONS DAST (diethylamino)sulfurtrifluoride DCM DichloromethaneDIEA diisopropylethylamine DMA Dimethylacetamide DMAP4-dimethylaminopyridine DME 1,2-dimethoxyethane DMFN,N-dimethylformamide DPPF 1,1′-bis(diphenylphosphino)ferrocene EDC1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EtOAc ethylacetate EtOH Ethanol HOAT Hydroxyazabenzotriazole K₂CO₃ Potassiumcarbonate MeCN Acetonitrile MgSO₄ Magnesium sulfate MeOH Methanol Na₂CO₃sodium carbonate NaCl Sodium chloride NaHCO₃ sodium bicarbonate NBSN-bromosuccinimide NMP N-methyl-2-pyrrolidone Pd₂(dba)₃Tris(dibenzylideneacetone)dipalladium(0) Pd(PPh₃)₄Tetrakis(triphenylphospine)palladium(0) Pd(dppf)Cl₂-Dichloro-(1,2-bis(diphenylphosphino)ethan)- DCMPalladium(II)-dichloromothethane adduct RT or rt room temperatureTBDMSCl tert-butyldimethylsilylchloride TEA Triethylamine THFtetrahydrofuran

Synthesis of 3-oxocyclohex-1-enyl trifluoromethanesulfonate

To a solution of cyclohexane-1,3-dione (1 equiv) in DCM (0.4 M) wasadded Na₂CO₃ (1.0 equiv.) and cooled to 0° C. Added Tf₂O (1.0 equiv.) inDCM (5 M) dropwise over 1 hr at room temperature under a nitrogenatmosphere. Upon addition, the reaction was stirred for 2 hr (dark redsolution). The solution was filtered and to the filtrate was addedsaturated NaHCO₃ (carefully), then extracted the organics, dried withbrine, then Na₂SO₄, and concentrated. The crude was used for the nextstep without further purification. 3-oxocyclohex-1-enyltrifluoromethanesulfonate was obtained in 67% yield. The triflatedecomposes upon storage and should be used immediately for the nextreaction. LC/MS=244.9/286.0 (M+H and M+CH₃CN); Rt=0.88 min.

Synthesis of3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone

To a solution of 3-oxocyclohex-1-enyl trifluoromethanesulfonate (1.0equiv.) in degassed dioxane (0.3 M) was added bis(pinacolato)diboron(2.0 equiv.), KOAc (3.0 equiv.), and Pd(dppf)Cl₂-DCM (0.05 equiv.). Thereaction was heated to 80° C. for 2 h, then filtered. The dioxanesolution was used for the next step without further purification.LC/MS=140.9 (M+H of boronic acid).

Synthesis of 3-(3-nitropyridin-4-yl)cyclohex-2-enone

To a solution of3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone (1.0equiv.) in degassed dioxane and 2M Na₂CO₃ was added4-chloro-3-nitropyridine (1.2 equiv.) and Pd(dppf)Cl₂-DCM (0.05 equiv.).The reaction was heated in an oil bath to 110° C. for 2 hours. Cooled toroom temperature, then diluted with EtOAc, added H₂O— dark solution,lots of emulsions. Filtered to get rid of the solids, then extracted theorganic phase, dried with Na₂SO₄, and concentrated. The crude waspurified via silica gel chromatography eluting with ethyl acetate andhexanes (1:1) to yield 3-(3-nitropyridin-4-yl)cyclohex-2-enone (55%, 2steps). LC/MS=219 (M+H), LC=2.294 min.

Synthesis of 3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 3-(3-nitropyridin-4-yl)cyclohex-2-enone (1.0 equiv.)was added EtOH (0.2 M) and CeCl₃-7H₂O (1.3 equiv.). The reaction wascooled to 0° C., then NaBH₄ (1.3 equiv.) was added in portions. Stirredfor 2 h at 0° C., then quenched by adding water, concentrated to removethe EtOH, added EtOAc, extracted the organics, dried with brine, thenNa₂SO₄, and concentrated to yield 3-(3-nitropyridin-4-yl)cyclohex-2-enol(99%). LC/MS=221.1 (M+H), LC=2.235 min.

Synthesis of2-(3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione

To a solution of 3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0 equiv.),triphenylphosphine (1.5 equiv.) and phthalimide (1.5 equiv.) in THF (0.3M) at 0° C. was added (E)-di-tert-butyl diazene-1,2-dicarboxylate (1.5equiv.) dropwise. The reaction was stirred at 0° C. for 2 hours.Concentrated to dryness under vacuo, then purified the crude via silicagel column chromatography eluting with EtOAc and hexanes (1:1 with 5%methanol) to afford the2-(3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione (63%yield). LC/MS=350.3 (M+H), LC=3.936 min.

Synthesis of2-(3-(3-aminopyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione

To a solution of2-(3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione (1.0equiv.) in AcOH (0.38 M) was added Fe (6.0 equiv.) and the reaction wasstirred at room temperature for 2 h. Filtered, then washed with methanoland concentrated the filtrate. To the crude was added ethyl acetate andsaturated NaHCO₃, the organics were dried with Na₂SO₄, and concentratedto give 2-(3-(3-aminopyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dioneas a yellow thick gum in 96% yield. LC/MS=320.0 (M+H), LC=2.410 min.

Synthesis of 2-(3-(3-aminopyridin-4-yl)cyclohexyl)isoindoline-1,3-dione

To a solution of2-(3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione (1.0equiv.) in acetic acid (0.1 M) was added 10% Pd/C (0.2 equiv.) and thereaction was stirred under a H₂ balloon. After 3 days, the reaction wasfiltered through Celite, washed with ethyl acetate and methanol, thefiltrate was diluted with ethyl acetate and washed twice with sat. 2MNa₂CO₃. The organic phase was dried with magnesium sulfate, filtered andconcentrated. The crude material was triturated with hexanes and ethylacetate to afford2-(3-(3-aminopyridin-4-yl)cyclohexyl)isoindoline-1,3-dione in 73% yield.LC/MS=322.2 (M+H), Rt=0.64 min.

Synthesis of 5,5-dimethyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate

In a 3-neck round-bottom flask, 5,5-dimethylcyclohexane-1,3-dione (1.0eq) was dissolved in DCM (0.2 M). Sodium carbonate (1.1 eq) was addedand the mixture was cooled with magnetic stirring on an ice/salt waterbath to ˜−5° C. under N₂. Triflic anhydride (1.05 equiv.) diluted in DCMwas added dropwise via addition funnel over 90 minutes. Upon completionof addition, the reaction was stirred at ˜0° C. for 1 h. From LCMS and¹H NMR, there was still starting material left. Additional sodiumcarbonate (0.51 eq) and triflic anhydride (0.50 eq) were added. After 2hours, the mixture was filtered through a coarse frit glass funnel (thecake was washed with DCM), transferred to an Erlenmeyer flask, quenchedby careful addition of saturated aqueous sodium bicarbonate withvigorous stirring until pH=7, transferred to a separatory funnel and thelayers separated. The organic layer was washed with brine, dried overMgSO₄, filtered and concentrated to give5,5-dimethyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate, which wasused to the next step without further purification. LC/MS (m/z):MH⁺=273.1, Rt=1.03.

Synthesis of5,5-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone

All of reagents 5,5-dimethyl-3-oxocyclohex-1-enyltrifluoromethanesulfonate (1.0 eq), potassium acetate (3.0 eq), andbis(pinacolato)diboron (2.0 eq) were added to 1,4-dioxane (0.2 M) in around bottomed flask and degassed by bubbling N₂ through the mixture for10 min. PdCl₂(dppf)-DCM adduct (0.03 eq) was added and the reactionheated to 80° C. fitted with a reflux condenser on an oil bath under N₂overnight. The mixture was cooled to room temperature, filtered througha coarse frit glass funnel, the cake rinsed with 1,4-dioxane to give the5,5-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enonein 1,4-dioxane which was used to next step without further purification.LC/MS (m/z): MH⁺(boronic acid)=169.1, Rt=0.50.

Synthesis of 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone

The boronate ester5,5-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone(1.0 eq) was dissolved in 1,4-dioxane in a round bottomed flask anddegassed by bubbling N₂ through the solution for 30 minutes.4-chloro-3-nitro-pyridine (1.3 eq) and 2M(aq) sodium carbonate (2.0 eq)were added and N₂ was bubbled through for 10 minutes and thenPdCl₂(dppf)-DCM (0.05 eq) was added. The reaction mixture was stirred at110° C. for 2 hr. The mixture was added to EtOAc and water. Theresulting mixture was filtered through celite, the cake was washed withEtOAc. The organic layer was separated and the aqueous was extractedwith EtOAc. The combined organic layers were washed with brine, driedover MgSO₄, filtered and concentrated. The residue was purified bysilica gel chromatography (eluted with EtOAc:Hexanes=1:10 to 2:1) togive 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone (46.7% forthree steps). LC/MS (m/z): MH⁺=247.2, Rt=0.79.

Synthesis of 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone(1.0 eq), and CeCl₃-7H₂O (1.2 eq) in MeOH (0.2 M) was added NaBH₄ (1.0eq) at 0° C. The solution was stirred for 1 hour, and then quenched bythe addition of 5 mL of water. The volatiles were removed in vacuum andthe residue was partitioned between EtOAc and H₂O. The organic layer wasseparated and washed with brine. The combined aqueous was back extractedwith EtOAc and the organic was washed with brine. The combined organicswere dried over MgSO₄, filtered and concentrated. The residue waspurified by column (5% methanol in 1:1 ethyl acetate and hexanes) togive 5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (74%). LC/MS(m/z): MH⁺=249.2, Rt=0.76.

Synthesis of2-(5,5-dimethyl-3-(3-nitropyridin-4-yl)-cyclohex-2-enyl)isoindoline-1,3-dione

To a homogeneous solution of5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0 eq), triphenylphosphine (1.5 eq), and phthalimide (1.5 eq) in THF (0.2 M) cooled to 0°C., ditertbutyl azodicarboxylate (1.5 eq) in THF was added to thesolution. The mixture was stirred at 0° C. for 2 hours. The reaction wasconcentrated in vacuo. The residue was purified by column (5% methanolin 1:1 ethyl acetate and hexanes) to give2-(5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione(99%). LC/MS (m/z): MH⁺=378.2, Rt=1.10.

Synthesis of2-(3-(3-aminopyridin-4-yl)-5,5-dimethyl-cyclohex-2-enyl)isoindoline-1,3-dione

A solution of2-(5,5-dimethyl-3-(3-nitropyridin-4-yl)cyclohex-2-enyl)isoindoline-1,3-dione(1 eq) in acetic acid (0.1M) was purged with nitrogen for 10 min. Then10% Pd/C (0.10 eq) was added. The reaction mixture was stirred at roomtemperature overnight under an atmosphere of hydrogen. Solids wereremoved by filtration over celite, then rinsed with EtOAc and MeOH. Thefiltrate was concentrated, diluted with EtOAc and washed 2× with sat.aq. 2M Na₂CO₃. The organic layer was dried with MgSO₄, filtered, andconcentrated. The residue was purified by column (5% methanol in 1:1ethyl acetate and hexanes) to give2-(3-(3-aminopyridin-4-yl)-5,5-dimethylcyclohex-2-enyl)isoindoline-1,3-dione(89%). LC/MS (m/z): MH⁺=348.3, Rt=0.79.

Synthesis of2-(5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dione

A solution of2-(3-(3-aminopyridin-4-yl)-5,5-dimethylcyclohex-2-enyl)isoindoline-1,3-dione(1.0 eq) in acetic acid (0.1M) was purged with nitrogen for 10 min. Then10% Pd/C (0.1 eq) was added. The reaction mixture was stirred at 45° C.,300 psi hydrogen atmosphere in a steel bomb overnight and at 65° C., 300psi for 5 hours. Solids were removed by filtration over celite, thenrinsed with EtOAc and MeOH. The filtrate was concentrated, diluted withEtOAc and washed 2× with sat. aq. 2M Na₂CO₃. The organic layer was driedwith MgSO₄, filtered, and concentrated. The residue was purified bycolumn (5% methanol in 1:1 ethyl acetate and hexanes) to give2-(5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dione(53%). LC/MS (m/z): MH⁺=350.3, Rt=0.78. The enantiomerically pure2-((1R,5R)-5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dioneand2-((1S,5S)-5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dionewere resolved by chiral HPLC (For analysis R_(t)=7.526 min and 13.105min respectively; hexanes:ethanol=80:20 (v:v), Chiralcel OJ-H 100×4.6 mmat 1 mL/min. For preparative separation, hexanes:ethanol=80:20 (v:v),Chiralcel OJ-H (250×20 mm at 20 mL/min). ¹H NMR (CDCl₃): δ 8.04 (s, 1H),8.00 (d, 1H), 7.82 (m, 2H), 7.71 (m, 2H), 7.06 (d, 1H), 4.54 (m, 1H),3.71 (m, 2H), 2.89 (m, 1H), 2.23-2.44 (m, 2H), 1.90 (m, 1H), 1.20-1.60(m, 3H), 1.18 (s, 3H), 1.07 (s, 3H).

Synthesis of 4-(cyclohexa-1,3-dienyl)-3-nitropyridine

To a solution of 3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0 equiv.) wasadded dioxane (0.18 M) and p-TSA (1.1 equiv.). The solution was heatedto 100° C. for 4 h. Cooled to room temperature, worked up with sat.NaHCO₃ and ethyl acetate, the organic phase was dried with Na₂SO₄ andconcentrated. The crude was purified via silica gel columnchromatography eluting with 100% DCM to give4-(cyclohexa-1,3-dienyl)-3-nitropyridine as a yellow oil (27% yield).LCMS (m/z): 203.1 (MH⁺), LC R_(t)=3.53 min, ¹H-NMR (CDCl₃): 9.02 (s,1H), 8.70 (d, J=5.3, 1H), 7.30 (d, J=5.3, 1H), 6.15-6.17 (m, 1H),6.02-6.11 (m, 2H), 2.35-2.38 (m, 4H).

Synthesis of (+/−)-2-azido-4-(3-nitropyridin-4-yl)cyclohex-3-enol

To a solution of 4-(cyclohexa-1,3-dienyl)-3-nitropyridine (1.0 equiv.)in DCM (0.1 M) was added NaHCO₃ (1.2 equiv.) to give a yellow solution.Cooled to 0° C., then added m-CPBA (1.0 equiv.) to the solution at onceas a solid. The reaction was stirred at 0° C. for 3.5 hr. Monitored byboth TLC and LC/MS. The product ionizes as M+H=237 (diol); Rt=0.41 minon UPLC. Quenched reaction with sat. NaHCO₃, then extracted with DCM (3times). The organic phase was further dried with brine, then Na₂SO₄,filtered and concentrated to give the crude epoxide as a yellow oil,which was used without further purification.

To a solution of the above crude material in EtOH and water (3:1)(cloudy yellow solution) was added NaN₃ (2.0 equiv.) and NH₄Cl (2.0equiv.) to give a clear orange solution. The reaction was stirred for 16h, then concentrated. EtOAc and water were added, the organic phase wasfurther dried with MgSO₄ and concentrated to give a brown oil. The oilwas loaded in silica gel and purified via column chromatography (ISCO,0-50% EtOAc) to give(+/−)-2-azido-4-(3-nitropyridin-4-yl)cyclohex-3-enol as a yellow oil(44% for 2 steps). LCMS (m/z)=262 (MH⁺), LC R_(t)=2.35 min.

Synthesis of(+/−)-4-(3-azido-4-(tert-butyldimethylsilyloxy)cyclohex-1-enyl)-3-nitropyridine

To a solution of (+/−)-2-azido-4-(3-nitropyridin-4-yl)cyclohex-3-enol(1.0 equiv.) in DCM (0.15 M) was added TBSCl (2.0 equiv.), imidazole(2.0 equiv.) and DMAP (0.1 equiv.) at room temperature. After 18 h,water was added, the organics were dried with brine, then Na₂SO₄, andconcentrated. The crude material was loaded to silica gel and purifiedvia column chromatography (ISCO) eluting with ethyl acetate and hexanes(20%). Obtained(+/−)-4-(3-azido-4-(tert-butyldimethylsilyloxy)cyclohex-1-enyl)-3-nitropyridineas a yellow oil in 60% yield. LCMS (m/z): 376.3 (MH⁺), LC R_(t)=5.848min.

Synthesis of (+/−)-tert-butyl6-(tert-butyldimethylsilyloxy)-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate

In a round-bottomed flask was added(+/−)-4-(3-azido-4-(tert-butyldimethylsilyloxy)cyclohex-1-enyl)-3-nitropyridine(1.0 equiv.) and pyridine (0.1 M) to give a yellow solution. Ammoniumhydroxide (10:1 pyridine: ammonium hydroxide) was added followed by PMe₃(3.0 equiv.). The reaction turned dark brown after 10 min. Stirred atroom temperature for 1.5 h. Quenched by adding EtOH, and concentrated.Repeated 2 more times. To the crude was added sat. NaHCO₃ and dioxane(1:1, 0.1M). Boc₂O (1.0 equiv.) was added. Stirred for one hour at roomtemperature. Washed with H₂O and EtOAc, the organic phase was dried withMgSO₄, filtered and concentrated. The residue was purified via silicagel column chromatography (ISCO, 5:1 Hex/EtOAc). Collected the purefractions and concentrated to give (+/−)-tert-butyl6-(tert-butyldimethylsilyloxy)-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamateas a foam. LCMS (m/z): 450.3 (MH⁺), LC R_(t)=5.83 min.

Synthesis of (+/−)-tert-butyl3-(3-aminopyridin-4-yl)-6-(tert-butyldimethylsilyloxy)cyclohex-2-enylcarbamate

To a solution of (+/−)-tert-butyl6-(tert-butyldimethylsilyloxy)-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate(1.0 equiv.) in AcOH (0.18 M) was added Fe (6.0 equiv.) and the reactionwas stirred for 20 h. Worked up by diluting the reaction with methanol,filtered, and concentrated the filtrate. To the crude was added ethylacetate and saturated NaHCO₃, the organics were dried with sodiumsulfate and concentrated to give (+/−)-tert-butyl3-(3-aminopyridin-4-yl)-6-(tert-butyldimethylsilyloxy)cyclohex-2-enylcarbamateas a yellow oil in 94% yield. LCMS (m/z): 420.3 (MH⁺), LC R_(t)=3.88min.

Synthesis of (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-2-(tert-butyldimethylsilyloxy)cyclohexylcarbamate

To a solution of (+/−)-tert-butyl3-(3-aminopyridin-4-yl)-6-(tert-butyldimethylsilyloxy)cyclohex-2-enylcarbamate(1.0 equiv.) in MeOH (0.1 M) was added Pd/C (20% by wt) and the reactionwas stirred under a hydrogen balloon for 18 h. LC/MS of the reactionindicated mixture of diastereomers, the reaction was filtered, washedwith EtOAc and concentrated the filtrate. The crude material waspurified via prep-HPLC (in DMSO), and the pure fractions were combined,neutralized with solid NaHCO₃, extracted with ethyl acetate, washed withbrine, dried under Na₂SO₄, and concentrated to give product A (8% yield)and product B (51% yield).

-   -   Product A: LCMS (m/z): 422.4 (MH⁺), LC R_(t)=3.75 min.    -   Product B: LCMS (m/z): 422.4 (MH⁺), LC R_(t)=3.94 min.

Synthesis of 1,4-dioxaspiro[4.5]dec-7-en-8-yl trifluoromethanesulfonate

1,4-Dioxaspiro[4.5]decan-8-one (1.0 equiv) was dissolved in Ether (0.1M)and stirred at −15° C. then 1M NaHMDS (1.05 equiv.) was added andstirred for 70 min then Tf₂O (1.05 equiv.) added and reaction allowed toslowly warm to rt. The mixture was stirred for 28 hr, washed with sat.aq. NaHCO₃ and then water. Aqueous layers combined and extracted withether. Organic layers combined, dried over MgSO₄, filtered, andconcentrated. The residue was purified by column (ethylether:hexanes=1:4) to give 1,4-dioxaspiro[4.5]dec-7-en-8-yltrifluoromethanesulfonate (65%). LC/MS (m/z): MH⁺=289.0, Rt=0.97. HPLCRt=3.77.

Synthesis of4,4,5,5-tetramethyl-2-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1,3,2-dioxaborolane

A solution of 1,4-dioxaspiro[4.5]dec-7-en-8-yl trifluoromethanesulfonate(1.0 equiv.) in dioxane (0.5 M) was purged with nitrogen for 30 min.Then 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.0equiv.), KOAc (3.0 equiv.), Pd(dppf)Cl₂-DCM (0.2 equiv.) were added andthe solution was stirred in a sealed bomb at 80° C. The reaction wasfiltered over a pad of celite, then to the filtrate was added ethylacetate, and washed with brine, dried over MgSO₄, filtered, andconcentrated. The residue was purified by column (ethylacetate:hexanes=1:1) to give4,4,5,5-tetramethyl-2-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1,3,2-dioxaborolane(95%). LC/MS (m/z): MH⁺=267.1, Rt=0.95.

Synthesis of 3-nitro-4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)pyridine

A solution of DME (0.2 M) and 2M aq. sodium carbonate (1.7 equiv.) waspurged with nitrogen for 20 min. Then 4-chloro-3-nitropyridine (1.6equiv.),4,4,5,5-tetramethyl-2-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1,3,2-dioxaborolane(1.0 equiv.), Pd(dppf)Cl₂-DCM (0.05 equiv.) were added and stirred in asealed bomb at 110° C. The reaction was stirred at that temperature for3.5 hours. The reaction was diluted with ethyl acetate, washed withwater, dried over MgSO₄, filtered, and concentrated. The residue waspurified by column (ethyl acetate:hexanes=1:1 with 10% methanol) to give3-nitro-4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)pyridine (83%). LC/MS (m/z):MH⁺=263.2, Rt=0.71.

Synthesis of 4-(3-nitropyridin-4-yl)cyclohex-3-enone

A mixture of 3-nitro-4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)pyridine (1.0equiv.) in 20% TFA in CH₂Cl₂ (0.2 M) was stirred at room temperatureovernight. The solvents were removed under reduced pressure. The residuewas dissolved with ethyl acetate (200 mL), and washed with sat NaHCO₃(30 mL), and sat NaCl (30 mL). The organic was dried with MgSO₄,filtered and concentrated to give4-(3-nitropyridin-4-yl)cyclohex-3-enone (85%). The crude product wasused to next step without further purification. LC/MS (m/z): MH⁺=218.9,Rt=0.60

Synthesis of 4-(3-nitropyridin-4-yl)cyclohex-3-enol

To a solution of 4-(3-nitropyridin-4-yl)cyclohex-3-enone (1.0 eq) inmethanol (0.2 M) was added sodium borohydride (1.8 equiv.) at 0° C. Thereaction mixture was stirred at 0° C. for 2 hr. Methanol was removedunder reduced pressure. The residue was dissolved with ethyl acetate(200 mL), and washed with sat. NaCl (30 mL). The organic was dried withMgSO₄, filtered and concentrated to give4-(3-nitropyridin-4-yl)cyclohex-3-enol (85%). The crude product was usedin the next step without further purification. LC/MS (m/z): MH⁺=221.0,Rt=0.55

Synthesis of 4-(3-nitropyridin-4-yl)cyclohex-3-enyl methanesulfonate

To a solution of 4-(3-nitropyridin-4-yl)cyclohex-3-enol (1.0 equiv.) andDIPEA (2.5 equiv.) in CH₂Cl₂ (0.15 M) was added methanesulfonyl chloride(1.8 equiv.) at 0° C. The reaction mixture was stirred at 0° C. for 1hr. The reaction mixture was diluted with ethyl acetate (200 mL), andwashed with sat NaCl (30 mL). The organic was dried MgSO₄, filtered andconcentrated to give 4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (93%). The residue was used in the next step withoutfurther purification. LC/MS (m/z): MH⁺=299.0, Rt=0.70

Synthesis of 4-(cyclohexa-1,3-dienyl)-3-nitropyridine

To a solution of 4-(3-nitropyridin-4-yl)cyclohex-3-enyl methanesulfonate(1.0 equiv) in tetrahydrofuran (0.1M) was added DBU (1.8 equiv.) at roomtemperature. The reaction mixture was stirred at rt overnight. Thereaction mixture was diluted with ethyl acetate (200 mL), and washedwith sat NaCl (30 mL). The organic was dried with MgSO₄, filtered andconcentrated. The residue was purified by column (5% methanol in 1:1ethyl acetate and hexanes) to give4-(cyclohexa-1,3-dienyl)-3-nitropyridine. LC/MS (m/z): MH⁺=203.2,Rt=0.85.

Synthesis of (+/−)-tert-butyl6-hydroxy-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate

To a solution of (+/−)-2-azido-4-(3-nitropyridin-4-yl)cyclohex-3-enol(1.0 equiv.) in Pyridine and NH₄OH (8:1, 0.23 M) was addedtrimethylphosphine (3.0 equiv.) at room temperature. The mixture wasstirred at room temperature for 3 hours. Solvents were removed. To theresidue was added ethanol. Then ethanol was removed in vacuo to ensureremoval of the ammonia totally. The residue was dissolved in 1,4-dioxaneand sat. aq. sodium bicarbonate, and then Boc₂O (1.0 eq) in THF wereadded to the mixture. The resulting mixture was stirred at roomtemperature for 2 hours. The reaction mixture was diluted with ethylacetate, and washed with sat NaCl. The organic was dried with MgSO₄,filtered and concentrated. The residue was purified by column (5%methanol in 1:1 ethyl acetate and hexanes) to give (+/−)-tert-butyl6-hydroxy-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (82%). LC/MS(m/z): MH⁺=336.0, Rt=0.71

Synthesis of(+/−)-2-(tert-butoxycarbonylamino)-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate

To a solution of (+/−)-tert-butyl6-hydroxy-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (1.0 equiv.)and triethyl amine (1.5 equiv.) in CH₂Cl₂ (0.2 M) was addedmethanesulfonyl chloride (1.2 equiv.) at 0° C. The mixture was stirredfor 2 hours at that temperature. The reaction mixture was diluted withethyl acetate, and washed with sat NaCl. The organic was dried withMgSO₄, filtered and concentrated to give(+/−)-2-(tert-butoxycarbonylamino)-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (85%), which was used in the next step without furtherpurification. LC/MS (m/z): MH⁺=414.0, Rt=0.82

Synthesis of(+/−)-5-(3-nitropyridin-4-yl)-3,3a,7,7a-tetrahydrobenzo[d]oxazol-2(6H)-one

The mixture of(+/−)-2-(tert-butoxycarbonylamino)-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (1.0 equiv.) in pyridine (0.21M) was stirred at 110° C.for 10 min in microwave. Pyridine was removed under reduced pressure.The residue was dissolved in ethyl acetate, and washed with sat NaCl.The organic was dried with MgSO₄, filtered and concentrated to give(+/−)-5-(3-nitropyridin-4-yl)-3,3a,7,7a-tetrahydrobenzo[d]oxazol-2(6H)-one(85%), which was used in the next step without further purification.LC/MS (m/z): MH⁺=262.1, Rt=0.49

Synthesis of (+/−)-tert-butyl5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate

To a solution of(+/−)-5-(3-nitropyridin-4-yl)-3,3a,7,7a-tetrahydrobenzo[d]oxazol-2(6H)-one(1.0 equiv.), TEA (1.8 equiv.), and catalytic amount DMAP in CH₂Cl₂(0.19 M) was added di-tert-butyl dicarbonate (1.2 eqiv) at roomtemperature. The reaction mixture was stirred for 1 hour. The reactionmixture was diluted with ethyl acetate (100 mL), and washed with satNaCl (30 mL). The organic was dried with MgSO₄, filtered andconcentrated. The residue was purified by column (5% methanol in 1:1ethyl acetate and hexanes) to give (+/−)-tert-butyl5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate(98%). LC/MS (m/z): MH⁺=306.0, Rt=0.75

Synthesis of (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-2-oxohexahydrobenzo[d]Oxazole-3(2H)-carboxylate

To a solution of (+/−)-tert-butyl5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate(1.0 equiv.) in methanol and ethyl acetate (1:1, 0.1 M) was added 10%Pd/C (0.1 equiv.). The resulting mixture was stirred under H₂ atmospherefor 6 hours. The solid was removed by filtration. The filtrate wasconcentrated under reduced pressure to give (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate(87%), which was used in the next step without further purification.LC/MS (m/z): MH⁺=334.1, Rt=0.51.

Synthesis of 5-methyl-3-oxocyclohex-1-enyltrifluoromethanesulfonate

To a solution of 5-methylcyclohexane-1,3-dione (1.0 equiv.) in DCM(0.5M) was added Na₂CO₃ (1.1 equiv.) and cooled to 0° C. Added Tf₂O (1.0equiv.) in DCM (5.0 M) dropwise over 1 hr at 0° C. under a nitrogenatmosphere. Upon addition, the reaction was stirred for 1 hr at roomtemperature (dark red solution). The solution was filtered and thefiltrate was quenched by careful addition of saturated NaHCO₃ withvigorous stirring until pH=7. The solution was transferred to aseparatory funnel and the layers were separated. The organic layer waswashed with brine, dried with Na₂SO₄, filtered, concentrated under vacuoand dried under high vacuum for 15 min to yield5-methyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate as light yellowoil in 78% yield. The triflate decomposes upon storage and should beused immediately for the next reaction. LC/MS=259.1/300.1 (M+H andM+CH₃CN); Rt=0.86 min, LC=3.84 min. ¹H-NMR (400 MHz, CDCl₃) δ ppm: 6.05(s, 1H), 2.70 (dd, J=17.2, 4.3, 1H), 2.53 (dd, J=16.6, 3.7, 1H),2.48-2.31 (m, 2H), 2.16 (dd, J=16.4, 11.7, 1H), 1.16 (d, J=5.9, 3H).

Synthesis of5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone

To a solution of 5-methyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate(1.0 equiv.) in degassed dioxane (0.7 M) was addedbis(pinacolato)diboron (2.0 equiv.), KOAc (3.0 equiv.), andPd(dppf)Cl₂-DCM (0.03 equiv.). The reaction was heated to 80° C. for 10h (initial heating at large scale results in exothermic formation of anorange foam on top of the solution, the heating bath should be removeduntil the foam retracts, reheating to 80° C. at this point appears to befine), then cooled to room temperature and filtered through a coarsefrit glass funnel. The cake was rinsed with more dioxane and thefiltrate solution was used for the next step without furtherpurification. LC/MS=155.1 (M+H of boronic acid); Rt=0.41 min, LC=1.37min.

Synthesis of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone

To a solution of5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-2-enone(1.0 equiv.) in degassed dioxane (0.5 M) and 2M Na₂CO₃ (2 equiv.) wasadded 4-chloro-3-nitropyridine (1.3 equiv.) and Pd(dppf)Cl₂-DCM (0.05equiv.). The reaction was placed under a reflux condenser and heated inan oil bath to 110° C. for 1 h. Cooled to room temperature, filteredthrough a pad of Celite, washed the pad with ethyl acetate andconcentrated the filtrate under vacuo. The residue was further pumped at80° C. on a rotary evaporator for one hour to remove boronateby-products (M+H=101) via sublimation. The residue was partitionedbetween brine and ethyl acetate, and the layers were separated, theaqueous phase was further extracted with ethyl acetate (4×), theorganics were combined, dried over sodium sulfate, filtered, andconcentrated. The crude was purified via silica gel chromatographyloading in DCM and eluting with 2-50% ethyl acetate and hexanes. Thepure fractions were concentrated in vacuo to yield an orange oil. Theoil was placed under high vacuum (˜500 mtorr) with seed crystalsovernight to yield an orange solid. The solid was further purified viatrituration in hexanes to yield5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone (48% 2 steps).LC/MS=233.2 (M+H); Rt=0.69 min, LC=2.70 min. ¹H-NMR (400 MHz, CdCl₃) δppm: 9.31 (s, 1H), 8.88 (d, J=5.1, 1H), 7.30 (d, J=5.1, 1H), 6.00 (d,J=2.4, 1H), 2.62 (dd, J=16.4, 3.5, 1H), 2.53-2.34 (m, 3H), 2.23 (dd,J=16.1, 11.7, 1H), 1.16 (d, J=6.3, 3H).

Synthesis of cis-(+/−)-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone (1.0equiv.) in EtOH (0.3 M) was added CeCl₃-7H₂O (1.2 equiv.). The reactionwas cooled to 0° C., then NaBH₄ (1.2 equiv.) was added in portions.Stirred for 1 h at 0° C., then quenched by adding water, concentrated toremove the EtOH, added EtOAc, extracted the organics, washed with brine,then dried with Na₂SO₄, filtered and concentrated to yieldcis-(+/−)-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (94%).LC/MS=235.2 (M+H), LC=2.62 min.

Synthesis of4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine

To a solution of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0equiv.) in DMF (0.5 M) was added imidazole (4.0 equiv.) and TBDMSC1 (2.5equiv.). After stirring for 18 hours the solution was portioned betweenEtOAc and H₂O and separated. After washing further with H₂O (3×) andNaCl_((sat.)), drying over MgSO₄, filtering and removal of solvents,4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridinewas obtained (85%). LC/MS=349.2 (M+H), LC=5.99 min.

Synthesis of4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)pyridin-3-amine

A heterogeneous solution of4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine(1.0 eq.) and iron (6.0 eq) in acetic acid, at a concentration of 0.4 M,was stirred vigorously for 2 hours. The mixture was then passed througha celite pad, eluting with MeOH. Upon removal of the volatiles in vacuo,the residue was dissolved in EtOAc, washed with Na₂CO_(3(sat.)),NaCl_((sat.)), was dried over MgSO₄, was filtered and the volatiles wereremoved in vacuo yielding4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)pyridin-3-amine(78%). LCMS (m/z): 319.3 (MH⁺); LC R_(t)=3.77 min.

Synthesis of4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine

To a solution of4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine(1.0 equiv.) in methanol, at a concentration of 0.1M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 15 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine(90%). LCMS (m/z): 321.3 (MH⁺); LC R_(t)=3.85 min.

Synthesis of cis (+/−) benzyl4-3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarbamate

To a solution ofcis-(+/−)-4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-aminein dichloromethane at a concentration of 0.5 M was added benzyl2,5-dioxopyrrolidin-1-yl carbonate (1.1 equiv.) and DMAP (0.05 equiv.).After stirring for 16 hours at rt, additional benzyl2,5-dioxopyrrolidin-1-yl carbonate (0.55 equiv.) and DMAP (0.03 equiv.)were added. After stirring for an additional 24 hours at rt, additionalbenzyl 2,5-dioxopyrrolidin-1-yl carbonate (0.1 equiv.) and DMAP (0.03equiv.) were added. After stirring for 18 more hours the solution waspartitioned between EtOAc and Na₂CO_(3(sat.)) and separated. Uponfurther washing with Na₂CO_(3(sat.)) (2×) and NaCl_((sat.)), drying overMgSO₄, filtering and removal of solvents, cis (+/−) benzyl4-3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarbamatewas obtained. The crude material was used as is. LC/MS=455.3 (M+H),LC=4.39 min.

Synthesis of cis-(+/−)benzyl4-(3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate

A solution of cis (+/−) benzyl4-3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarbamatein 1:2:1 6N HCl/THF/MeOH at a concentration of 0.1M was stirred at rtfor 6 hours. The pH was than adjusted to pH=7 by addition of 6N NaOH andthe volatiles were removed in vacuo. The aqueous layer was extractedwith EtOAc and the organic was washed with NaCl_((sat.)), dried overMgSO₄, filtered and upon removal of the volatiles in vacuo,cis-(+/−)benzyl 4-(3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamatewas obtained. The crude material was used as is. LC/MS=341.2 (M+H),LC=2.38 min.

Synthesis of cis (+/−)-benzyl4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate

To a 0° C. solution of cis-(+/−)-benzyl4-(3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate in wet CH₂Cl₂ at aconcentration of 0.16 M was added Dess-Martin Periodinane (1.5 equiv.)and the solution was stirred for 18 hours as it warmed to rt. Thesolution was partitioned between EtOAc and 1:1 10%Na₂S₂O₃/NaHCO_(3(sat.)) and separated. Upon further washing with 1:1 10%Na₂S₂O₃/NaHCO_(3(sat.)) (2×) and NaCl_((sat.)), drying over MgSO₄,filtering, removal of solvents and purification by silica gelchromatography (75-100% EtOAc/hexanes),cis-(+/−)-benzyl-4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate wasobtained as a white solid (53%, 5 steps). LC/MS=339.2 (M+H).

Synthesis of cis-(+/−)-benzyl4-(−3-(benzylamino)-5-methylcyclohexyl)pyridin-3-ylcarbamate

A solution ofcis-(+/−)-benzyl-4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate (1.0equiv) and benzylamine (3.0 equiv) in MeOH, at a concentration of 0.25M, was stirred at rt for 2 hours. Upon cooling in a −78° C. bath, LiBH₄(1.1 equiv, 2.0 M in THF) was added and the solution was allowed to warmto rt with stirring over 16 hours. The solution was partitioned betweenEtOAc and NaHCO_(3(sat.)), separated, washed further withNaHCO_(3(sat.)) and NaCl_((sat.)), dried over MgSO₄, filtered and afterremoval of volatiles in vacuo, cis-(+/−)-benzyl4-(-3-(benzylamino)-5-methylcyclohexyl)pyridin-3-ylcarbamate wasobtained as a 4:1 mixture of isomers, with the all cis as predominantLC/MS=430.3 (M+H), LC=0.62 min.

Synthesis of cis(+/−)-tert-butyl(-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate

To a solution of cis-(+/−)-benzyl4-(-3-(benzylamino)-5-methylcyclohexyl)pyridin-3-ylcarbamate was (1.0equiv.) in methanol, at a concentration of 0.07 M, was added 20%palladium hydroxide on carbon (0.2 eq.). The resultant heterogeneoussolution was put under an atmosphere of hydrogen and was stirred for 14hours. At this time the reaction was purged with Ar, Boc₂O (1.0 equiv.)was added and the solution was stirred for 8 hours. Additional Boc₂O(1.0 equiv.) was added and the solution was stirred for 16 more hours.At this time the mixture was filtered through a pad of celite elutingwith methanol. Upon removal of volatiles in vacuo, purification bysilica gel chromatography (2.5-2.5 MeOH/CH₂Cl₂ with 0.1% DIEA) andrecrystallization from 10% EtOAc/hexanes yielded cis (+/−)-tert-butyl(-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate (49%). LCMS (m/z):306.3 (MH⁺), LC R_(t)=2.59 min. Pure enantiomers could be obtained bychiral chromatography.

Synthesis of (+/−)-4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine

To a solution of (+/−)-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol(1.0 equiv.) in dioxane (0.1M) was added p-TSA (1.0 equiv.), and thereaction was stirred at 100° C. for 3 h. The solution was cooled to roomtemperature, then passed through a pad of neutral alumina eluting withEtOAc to yield (+/−)-4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine asa yellow oil in 68% yield. LC/MS=217.1 (M+H), LC=3.908 min.

Synthesis of(+/−)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol

To a solution of (+/−)-4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine(1.0 equiv.) in DCM (0.1M) at 0° C. was added m-CPBA (1.1 equiv.) andthe reaction was allowed to warm to room temperature. After 3 hours, themixture was quenched with saturated NaHCO₃, extracted with DCM, and theorganic phase was dried with sodium sulfate, filtered, and concentratedto give a yellow oil. The crude was dissolved in ethanol and water (3:1,0.1 M), and sodium azide (2.0 equiv.) and ammonium chloride (2.0 equiv.)were added. The reaction was stirred for 4 hours, then concentrated invacuo. To the crude was added ethyl acetate and water, the organic phasewas washed with brine, dried with sodium sulfate, filtered, andconcentrated. The crude material was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to afford(+/−)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol as an oilin 49% yield. LC/MS=276.1 (M+H), Rt=0.71 min.

Synthesis of tert-butyl(+/−)-6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate

To a solution of(+/−)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol (1.0equiv.) in pyridine and ammonium hydroxide (8:1, 0.08 M) was addedtrimethylphosphine (3.0 equiv.) and the brown solution was stirred atroom temperature for 2 hours. Ethanol was added to the mixture and thesolution was concentrated under vacuo (2×). The crude mixture was thendissolved in dioxane and sat. NaHCO₃ (1:1, 0.08 M) and Boc₂O (1.0equiv.) was added. The solution was stirred at room temperature for 2hours, then partitioned between ethyl acetate and water. The organicphase was dried with magnesium sulfate, filtered, and concentrated invacuo. The crude product was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to affordtert-butyl(+/−)-6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamatein 69% yield. LC/MS=350.1 (M+H), Rt=0.76 min.

Synthesis of(+/−)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enyl methanesulfonate

To a solution of tert-butyl(+/−)-6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate(1.0 equiv.) in DCM (0.09 M) was added triethyl amine (1.5 equiv.). Thereaction mixture was cooled to 0° C. and MsCl (1.2 equiv.) was added tothe reaction and stirred for 3 hours. To the solution was added water,the organic phase was dried with sodium sulfate, filtered, andconcentrated. The crude material was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to give(+/−)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate as a white foam in 65% yield. LC/MS=428.2 (M+H),Rt=0.88 min.

Synthesis of (+/−)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate

A solution of(+/−)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (1.0 equiv.) in pyridine (0.2 M) in a microwave vesselwas heated to 110° C. for 10 min. The orange solution was thenconcentrated to dryness and worked up by partitioning between ethylacetate and water. The organic phase was dried with sodium sulfate,filtered and concentrated. The crude material was dissolved in DCM (0.2M) and triethyl amine (1.8 equiv.) was added to the reaction, followedby Boc₂O (1.2 eqiv.). After stirring at room temperature for 40 min, thereaction was concentrated in vacuo and purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to give(+/−)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylateas a white foam in 66% yield. LC/MS=376.0 (M+H), Rt=0.87 min.

Synthesis of (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate

To a solution of (+/−)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate(1.0 equiv.) in MeOH and ethyl acetate (1:1, 0.07 M) was added 10% Pd/C(0.1 equiv.) and the reaction was stirred at room temperature under anatmosphere of hydrogen. Upon completion of the reaction, the solutionwas filtered through a pad of Celite, washed with MeOH and ethylacetate, the filtrate was concentrated to dryness under vacuo to give(+/−)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylateas a mixture of diastereomers in >99% yield. LC/MS=348.2 (M+H), Rt=0.50min.

Synthesis of(+/−)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol

To a solution of 4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine (1.0equiv.) in THF and water (1:1, 0.13 M) was added NBS (1.5 equiv.) andthe reaction was stirred at room temperature for 30 min. Uponcompletion, ethyl acetate and water were added to the reaction, theorganic phase was dried with brine, then sodium sulfate, filtered, andconcentrated. The crude material was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to give(+/−)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol as ayellow oil in 80% yield. LC/MS=315.0/313.0 (M+H), LC=2.966 min.

Synthesis of(+/−)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol

To a solution of(+/−)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (1.0equiv.) in THF (0.1M) was added potassium tert-butoxide (1.5 equiv.).The reaction turned from orange to black almost immediately. By TLC, theformation of product is clean in 30 min. Quenched by adding saturatedammonium chloride and ethyl acetate. The organic phase was dried withbrine, then sodium sulfate, filtered, and concentrated. The crudeproduct was dissolved in ethanol and water (3:1, 0.1M), and ammoniumchloride (2.0 equiv) and sodium azide (2.0 equiv.) were added. The darkorange reaction was stirred at room temperature overnight. Theconversion to product is clean as indicated by LC/MS. The reaction wasconcentrated to remove the ethanol, ethyl acetate and water were added,and the organic phase was dried with sodium sulfate, filtered, andconcentrated. The crude material was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to give(+/−)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol in 55%yield. LC/MS=276.0 (M+H), LC=2.803 min.

Synthesis of (+/−)-tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate

To a solution of(+/−)-2-azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol (1.0equiv.) in pyridine and ammonium hydroxide (8:1, 0.08 M) was addedtrimethylphosphine (3.0 equiv.) and the brown solution was stirred atroom temperature for 2 h. Upon completion, EtOH was added and thesolution was concentrated in vacuo. More ethanol was added and thereaction was concentrated again. Dioxane and sat. NaHCO₃ (1:1, 0.08 M)were added to the crude, followed by Boc₂O (1.0 equiv.). Stirred thereaction mixture at room temperature for 2 h, then added water and ethylacetate. The organic phase was dried with MgSO₄, and concentrated. Thecrude product was purified via silica gel column chromatography elutingwith ethyl acetate and hexanes (1:1) to afford (+/−)-tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate(59%). LC/MS=350.1 (M+H), Rt: 0.76 min.

Synthesis of(+/−)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylacetate

To a solution of (+/−)-tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (1.0equiv.) in pyridine (0.1 M) was added Ac₂O (2.0 equiv.) and the reactionwas stirred at room temperature overnight. Upon completion, the reactionwas concentrated to dryness, then worked-up with ethyl acetate andwater. The organic phase was dried with brine, then sodium sulfate,filtered, and concentrated to give(+/−)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylacetate in 94% yield. LC/MS=392.2 (M+H), Rt=0.94 min.

Synthesis of(+/−)-4-(3-aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexylacetate

To a degassed solution of(+/−)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylacetate (1.0 equiv.) in MeOH and EtOAc (1:1, 0.1M) was added 10% Pd/C(0.1 equiv.) and the reaction was stirred at room temperature under ahydrogen balloon for 3 days. Upon completion, the solution was filteredthrough a pad of Celite, the pad was washed with ethyl acetate and thefiltrate was concentrated. The crude material contained about 10% of theundesired isomer. The crude was dissolved in ethyl acetate (˜20%) andhexanes and heated until all dissolved. The solution was allowed to sitat room temperature for 2 days. The precipitate was then collected togive(+/−)-4-(3-aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexylacetate as the pure product in 59% yield. LC/MS=364.3 (M+H), Rt=0.63min.

Synthesis of2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate

To a solution of tert-butyl6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enylcarbamate (1.0equiv.) in DCM (0.09 M) was added triethylamine (1.5 equiv.) and thereaction was cooled to 0° C. MsCl (1.2 equiv.) was added to the reactionand stirred for 3 h. Another 1.0 equiv. of MsCl was added to thereaction and stirred for another 2 h. Worked up the reaction by addingwater, the organic phase was dried with brine, sodium sulfate, andconcentrated. The crude product was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:1) to afford2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate as a white foam in 65% yield. LC/MS=428.2 (M+H), LC:3.542 min.

Synthesis of (+/−)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate

A solution of(+/−)-2-(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enylmethanesulfonate (1.0 equiv.) in pyridine (0.2 M) was heated in themicrowave at 110° C. for 10 min. The orange reaction was thenconcentrated under vacuo, the crude was dissolved in ethyl acetate andwater, the organic phase was dried with sodium sulfate and concentratedunder vacuo. The crude material was dissolved in DCM (0.2 M),triethylamine (1.8 equiv.) was added, followed by Boc₂O (1.2 equiv.).The reaction was stirred for 40 min, then concentrated to dryness. Thecrude material was purified via silica gel column chromatography elutingwith hexane and ethyl acetate (1:1) to afford (+/−)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylateas a white foam in 66% yield. LC/MS=376.0 (M+H), LC: 3.424 min.

Synthesis of (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate

To a degassed solution of (+/−)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxo-3a,6,7,7a-tetrahydrobenzo[d]oxazole-3(2H)-carboxylate(1.0 equiv.) in MeOH and EtOAc (1:1, 0.1M) was added 10% Pd/C (0.1equiv.). The reaction was stirred under a hydrogen balloon overnight.Upon completion, the solution was filtered through a pad of Celite andthe pad was washed with ethyl acetate. The filtrate was concentratedunder vacuo to give (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylateas the desired product as a yellow foam in 93% yield. LC/MS=348.1 (M+H),Rt=055 min.

Synthesis of((+/−)-(1R,2R,6S)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-dioland(+/−)-(1R,2S,6S)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-diol)

To a solution of(+/−)-(1S,5S,6S)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol(1.0 equiv.) in THF (0.1M) was added potassium tert-butoxide (1.5equiv.) at room temperature. The reaction mixture was stirred for 10min. The reaction mixture was quenched with NH₄Cl solution and worked upwith EtOAc by washing with water and brine. The organic layer was driedover anhydrous sodium sulfate, filtered off, and dried in vacuo. Thecrude product was used for next step without further purification.R_(f)=0.5 (50% EtOAc/Hexanes). LCMS: MH+251.2 (as a diol), R_(t)=0.49min. To a solution of crude(+/−)-4-((1S,5S)-5-methyl-7-oxabicyclo[4.1.0]hept-2-en-3-yl)-3-nitropyridine(1.0 equiv.) in 2:1 CH₃CN/H₂O (0.1 M) was added acetic acid (0.3 equiv.)at room temperature. The reaction mixture was stirred for 16 h at roomtemperature. After quenched with NaHCO₃ solution, the reaction mixturewas concentrated to remove the majority of CH₃CN and the residue waspartitioned between EtOAc and water. The combined organic layer waswashed with water and brine, dried over anhydrous sodium sulfate,filtered and concentrated in vacuo. A mixture of diols((+/−)-(1R,2R,6S)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-dioland(+/−)-(1R,2S,6S)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-diol)was obtained in 33.1% yield as a white solid by flash columnchromatography. R_(f)=0.3 (100% EtOAc; diols were not separable on TLC).LCMS: MH+251.2, R_(t)=0.49 min.

Synthesis of(+/−)-4-((3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridineand(+/−)-4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine

To a solution of a mixture of diols (1.0 equiv) in DMF (0.3M) was addedTBDMSC1 (7.0 equiv.) and imidazole (9 equiv.) at room temperature. Thereaction mixture was stirred at room temperature overnight. Afterquenched with sat NaHCO₃, The reaction mixture was extracted with EtOAc.The combined organic layer was washed with water and brine, dried overanhydrous sodium sulfate, filtered and concentrated in vacuo. Themixture was purified by sequential automated silica columnchromatography (gradient eluting with EtOAc and Hexanes) and preparativereverse phase HPLC (55%-95% acetonitrile in water, then run 5%-95%acetonitrile in water to yield(+/−)-4-((3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine(27.2%) and(+/−)-4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine(50.2%). LCMS: MH+479.2, R_(t)=1.60 and 1.63 min.

Synthesis of4-((1S,3S,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amineand of4-((1R,3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine

To a solution of(+/−)-4-((3R,4R,5S)-3,4-bis(tert-butyldimethyl-silyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine(1.0 equiv.) in ethanol/EtOAc, at a concentration of 0.1 M, was added10% palladium on carbon (0.1 eq.). The resultant heterogeneous solutionwas put under an atmosphere of hydrogen and was stirred for 14 hours. Atthis time the mixture was filtered through a pad of celite eluting withEtOAc. The volatiles were removed in vacuo and the crude material waspurified by automated silica column chromatography (R_(f)=0.2, 40% EtOAcin Heptane) to yield pure racemic product. LCMS: MH+451.3, Rt=1.35 min.The racemic compound was resolved by chiral chromatography (IC column, 1mL/min, 5% IPA in Heptane) to yield4-((1S,3S,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine(6.01 min) and4-((1R,3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine(8.34 min)

Synthesis of4-((1R,3R,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amineand of4-((1S,3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine

To a solution of(+/−)-4-((3S,4R,5S)-3,4-bis(tert-butyldimethyl-silyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine(1.0 equiv.) in ethanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 14 hours. Atthis time the mixture was filtered through a pad of celite eluting withethanol. The volatiles were removed in vacuo and the crude material waspurified by automated silica column chromatography (R_(f)=0.2, 40% EtOAcin Heptane) to yield pure racemic product (50.4%). LCMS: MH+451.3,R_(t)=1.35 min. The racemic compound was resolved by chiralchromatography (IC column, 1 mL/min, 5% IPA in Heptane) to yield4-((1R,3R,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine(6.98 min) and4-((1S,3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methyl-cyclohexyl)pyridin-3-amine(8.67 min)

Synthesis of sodium6-(methoxycarbonyl)-3-oxo-5-(trifluoromethyl)cyclohex-1-enolate

To a freshly prepared solution of sodium (1.0 eq) in t-BuOH (1M) wasadded ethyl acetoacetate (1.0 eq) by dropwise and the mixture stirred onan ice bath for an additional 15 min. ethyl 4,4,4-trifluorocrotonate(1.0 eq) was added dropwise and the mixture stirred at room temperaturefor an additional 30 min. After refluxing for 2 h, the mixture wascooled and hexanes was added. The precipitate was filtered withoutfurther purification to give sodium6-(methoxycarbonyl)-3-oxo-5-(trifluoromethyl)cyclohex-1-enolate (46%).LC/MS (m/z): MH⁺=253.1, Rt=0.70 min.

Synthesis of 5-(trifluoromethyl)cyclohexane-1,3-dione

Sodium 6-(Methoxycarbonyl)-3-oxo-5-(trifluoromethyl)cyclohex-1-enolate(1.0 eq) was dissolved in 1M NaOH (1.0 eq), and the mixture refluxed for1 h. After cooling to room temperature, the mixture was acidified with 5M sulfuric acid. The mixture was extracted with EtOAc. After washingwith water, the organic layer was dried over magnesium sulfate, thesolvent was removed under reduced pressure to give5-(trifluoromethyl)cyclohexane-1,3-dione, which was used to the nextstep without further purification (98%). LC/MS (m/z): MH⁺=181.1, Rt=0.55min.

Synthesis of 3-oxo-5-(trifluoromethyl)cyclohex-1-enyltrifluoromethanesulfonate

To a suspension of 5-(trifluoromethyl)cyclohexane-1,3-dione (1.0 eq) inDCM (0.23 M) was added TEA (1.2 eq) to give a clear solution. Themixture was cooled to 0° C. And then Tf₂O (1.05 eq) in DCM was addeddropwise. The reaction mixture was stirred at that temperature for 2hours. The reaction mixture was diluted with DCM, and washed with water,aq. NaHCO₃, brine, and was dried over MgSO₄, filtered and concentratedto give 3-oxo-5-(trifluoromethyl)cyclohex-1-enyltrifluoromethanesulfonate, which was used to next step directly. LC/MS(m/z): MH⁺=313.0, Rt=1.02 min.

Synthesis of3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)cyclohex-2-enone

All of reagents 3-oxo-5-(trifluoromethyl)cyclohex-1-enyltrifluoromethanesulfonate (1.0 eq), NaOAc (3.0 eq), andbis(pinacolato)diboron (2.0 eq) were added to 1,4-dioxane (0.23 M) in around bottom flask and degassed by bubbling N₂ through the mixture for10 min. PdCl₂(dppf).CH₂Cl₂ adduct (0.1 eq) was added and the reactionheated to 80° C. fitted with a reflux condenser on an oil bath under N₂for two hours. The mixture was cooled to room temperature, filteredthrough a coarse frit glass funnel, the cake rinsed with ˜10 mL1,4-dioxane to give3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)cyclohex-2-enonein 1,4-dioxane, which was used to next step directly. LC/MS (m/z):MH⁺=209.1 (boronic acid), Rt=0.60 min.

Synthesis of 3-(3-nitropyridin-4-yl)-5-(trifluoromethyl)cyclohex-2-enone

The boronate ester3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)cyclohex-2-enone(1.0 eq) was dissolved in 1,4-dioxane (0.14 M) in a round bottom flaskwas degassed by bubbling N₂ through the solution for 30 minutes.4-chloro-3-nitropyridine (1.3 eq) and aq. Na₂CO₃ (2M, 2.0 eq) were addedand N₂ was bubbled through for 10 minutes and then PdCl₂(dppf).CH₂Cl₂adduct (0.1 eq) was added. The reaction mixture was stirred at 100° C.for 2 Hours. The mixture was added EtOAc and brine. The resultingmixture was filtered through celite, the cake was washed with EtOAc. Theorganic layer was separated, and washed with brine, dried over MgSO4,and filtered and concentrated. The crude product was purified by silicagel chromatography (eluted with EtOAc:Hexanes=1:10 to 2:1) to give3-(3-nitropyridin-4-yl)-5-(trifluoromethyl)cyclohex-2-enone (73% forthree steps from diketone). LC/MS (m/z): MH⁺=287.1, Rt=0.85 min.

Synthesis ofcis-3-(3-nitropyridin-4-yl)-5-(trifluoromethyl)cyclohex-2-enol

3-(3-Nitropyridin-4-yl)-5-(trifluoromethyl)cyclohex-2-enone (1.0 eq) wasmixed with cerium(III) chloride heptahydrate (1.0 eq) and absoluteethanol (0.17) was added. The mixture was stirred at ambient temperatureuntil all solids dissolved. the mixture was cooled on an ice bath andNaBH₄ (1.2 eq) was added portion wise. The reaction was stirred on theice bath for 1 h. The mixture was diluted with EtOAc, washed with water,dried over MgSO₄, filtered and concentrated. The residue was purified bycolumn (1:1 ethyl acetate and hexanes) to givecis-3-(3-nitropyridin-4-yl)-5-(trifluoromethyl)cyclohex-2-enol (66%).LC/MS (m/z): MH⁺=289.2, Rt=0.72 min.

Synthesis ofcis-4-(3-azido-5-(trifluoromethyl)cyclohex-1-enyl)-3-nitropyridine

To a solution ofcis-3-(3-nitropyridin-4-yl)-5-(trifluoromethyl)cyclohex-2-enol (1.0 eq)in DCM (0.14 M) was added TEA (2.5 eq), and followed by MsCl (1.8 eq) atroom temperature. The reaction mixture was stirred at room temperaturefor 2 hours. The solvent was removed. The residue was dissolved in DMF(0.19 M), and then the mixture was added sodium azide (1.2 eq). Theresulting mixture was stirred at room temperature for 1 hour. Another1.2 eq of sodium azide was added. The mixture was stirred at roomtemperature overnight. The reaction mixture was diluted with ethylacetate and heptane, and washed with sat NaCl. The organic was driedover MgSO₄, filtered and concentrated. The residue was purified bycolumn (1:1 ethyl acetate and hexanes) to givecis-4-(3-azido-5-(trifluoromethyl)cyclohex-1-enyl)-3-nitropyridine(58%). LC/MS (m/z): MH⁺=314.1, Rt=0.96 min.

Synthesis of tert-butyl(1R,3R,5S)-3-(3-aminopyridin-4-yl)-5-(trifluoromethyl)cyclohexylcarbamate

A solution ofcis-4-(3-azido-5-(trifluoromethyl)cyclohex-1-enyl)-3-nitropyridine (1.0eq) in Ethanol (0.13 M) was bubbled with N₂ for 20 min. Then thereaction mixture was added Boc-anhydride (1.5 eq) and Pd/C (0.2 eq). Thereaction mixture was stirred at room temperature under H₂ atmosphereovernight. Solid was removed by filtration over celite and rinsed withEtOH. The residue was purified by column (5% methanol in 1:1 ethylacetate and hexanes) to give racemiccis-3-(3-aminopyridin-4-yl)-5-(trifluoromethyl)cyclohexylcarbamate(57%). LC/MS (m/z): MH+=360.2, Rt=0.72 min. The enantiomerically puretert-butyl(1R,3R,5S)-3-(3-aminopyridin-4-yl)-5-(trifluoromethyl)cyclohexylcarbamateandN-(4-((1S,3S,5R)-3-amino-5-(trifluoromethyl)cyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamidewere resolved by chiral HPLC (For analysis R_(t)=8.14 min and 10.59 minrespectively; heptane:isopropanol=90:10 (v:v), Chiralcel IC 100×4 6 mmat 1 mL/min. For preparative separation, heptane:isopropanol=90:10(v:v), Chiralcel IC 250×20 mm at 15 mL/min)

Synthesis of(R)-4-benzyl-3-((2R,3R)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3-hydroxy-2-methylpropanoyl)oxazolidin-2-one

(R)-4-benzyl-3-((2R,3R)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3-hydroxy-2-methylpropanoyl)oxazolidin-2-onewas prepared in the reported manner (Proc.Nat.Acad.Sciences, 101, 33,2004, pages 12042-12047) for the enantiomeric compound by starting with(R)-4-benzyl-3-propionyloxazolidin-2-one and R-glyceraldehyde acetonide.

Synthesis of(R)-4-benzyl-3-((2R,3R)-3-(tert-butyldimethylsilyloxy)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropanoyl)oxazolidin-2-one

(R)-4-benzyl-3-((2R,3R)-3-(tert-butyldimethylsilyloxy)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropanoyl)oxazolidin-2-onewas prepared in the reported manner (Proc.Nat.Acad.Sciences, 101, 33,2004, pages 12042-12047) for the enantiomeric compound by starting with(R)-4-benzyl-3-((2R,3R)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-3-hydroxy-2-methylpropanoyl)oxazolidin-2-one.

Synthesis of(2S,3R)-3-(tert-butyldimethylsilyloxy)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropan-1-ol

To a solution of(R)-4-benzyl-3-((2R,3R)-3-(tert-butyldimethylsilyloxy)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropanoyl)oxazolidin-2-one(1.0 equiv.) and ethanol (3.0 equiv.) in THF (0.09 M) was added LiBH₄(1.0 equiv.) at −40° C. The reaction mixture was allowed to warm up tort slowly and stirred at that temperature for 12 hours. The solution wascooled back to −40° C. and additional LiBH₄ (0.3 equiv.) was added.After warming back up to rt and stirring for 2 hours the solution wasthen diluted with diethyl ether and 1N NaOH) was added. The resultingmixture was extracted with ethyl acetate, the organic layer wasseparated, washed with NaCl_((sat.)), dried over magnesium sulfate,filtered, and concentrated. The residue was purified via silica gelcolumn chromatography (10-30% EtOAc/n-heptanes) yielding(2S,3R)-3-(tert-butyldimethylsilyloxy)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropan-1-olin (75%). LC/MS=247.1 (M+H-ketal-H₂O), R_(t)=0.64 min.

Synthesis of a((1R,2S)-3-azido-1-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropoxy)(tert-butyl)dimethylsilane

To a solution of(2S,3R)-3-(tert-butyldimethylsilyloxy)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropan-1-ol(1.0 equiv.), DIAD (2.0 equiv.), and PPh₃ (2.0 equiv.) in THF (0.18 M)was added DPPA (1.0 equiv., 1M solution in THF). The reaction mixturewas stirred at room temperature overnight. Upon removal of the volatilesunder vacuo, the residue was purified by silica gel columnchromatography (2-3-5% EtOAc/n-heptanes) yielding((1R,2S)-3-azido-1-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropoxy)(tert-butyl)dimethylsilane(62%). ¹H-NMR (400 MHz, CDCl₃): δ 4.04-4.10 (m, 1H), 3.94 (dd, J=8.0,6.4, 1H), 3.72 (d, J=7.2, 1H), 3.53 (t, J=8.0, 1H), 3.36 (dd, J=12, 8.0,1H), 3.19 (dd, J=12.0, 6.7, 1H), 1.52-1.60 (m, 1H), 1.41 (s, 3H), 1.34(s, 3H), 0.92 (d, J=7.2, 3H), 0.90 (s, 9H), 0.12 (s, 3H), 0.09 (s, 3H).

Synthesis of(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-4-methylpentane-1,2-diol

To a solution of((1R,2S)-3-azido-1-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylpropoxy)(tert-butyl)dimethylsilane(1.0 equiv.) in MeOH (0.1 M) was added PPTS (1.0 equiv.) and the mixturewas stirred at rt for 14 hours, 50° C. for 2 hours and 80° C. for 1hour. The volatiles were removed under vacuo and the residue waspurified via silica gel column chromatography (10-25% EtOAc/n-heptanes)yielding(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-4-methylpentane-1,2-diol(40%).

Synthesis of(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-2-hydroxy-4-methylpentyl4-methylbenzenesulfonate

To a solution of tert-butyl(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-1-hydroxy-4-methylpentan-2-ylcarbamate(1.0 equiv.) in pyridine (0.2 M) was added pTsCl (1.3 equiv.) at 0° C.The mixture held at this temperature for 16 hours. The volatiles wereremoved in vacuo and the residue was purified by silica gel columnchromatography (10-15-20% EtOAc/n-heptanes) yielding(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-2-hydroxy-4-methylpentyl4-methylbenzenesulfonate.

Synthesis of(2R,3R,4S)-5-azido-2,3-bis(tert-butyldimethylsilyloxy)-4-methylpentyl4-methylbenzenesulfonate

To a solution of(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-2-hydroxy-4-methylpentyl4-methylbenzenesulfonate (1.0 equiv.) and 2,6-lutidine (3.4 equiv.) wasadded TBDMSOTf (1.7 equiv.) at 0° C. The solution was stirred for 7hours as it warmed to rt. The solution was diluted with EtOAc, washedwith 10% CuSO₄, H₂O, Na₂CO_(3(sat.)), NaCl_((sat.)), dried over MgSO₄,filtered and concentrated. The residue was purified by silica gel columnchromatography (2.5-5-10-20% EtOAc/n-heptanes) yielding(2R,3R,4S)-5-azido-2,3-bis(tert-butyldimethylsilyloxy)-4-methylpentyl4-methylbenzenesulfonate (75%).

Synthesis of4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylpiperidin-1-yl)-3-nitropyridine

A solution of(2R,3R,4S)-5-azido-2,3-bis(tert-butyldimethylsilyloxy)-4-methylpentyl4-methylbenzenesulfonate in EtOH (0.05 M) was degassed with argon. DIEA(1.5 equiv.) was added, followed by 10% Pd/C (0.1 equiv.). The reactionmixture was stirred under a hydrogen balloon for 3 hours. The solutionwas degassed and purged to argon, at which time 4-chloro-3-nitropyridine(1.5 equiv.) and additional DIEA (1.5 equiv.) were added. After stirringat rt for 15 hours the solution was filtered to remove the Pd/C and thevolatiles were removed in vacuo. The residue was diluted with ethylacetate and washed with Na₂CO_(3(sat.)), NaCl_((sat.)), dried overMgSO₄, filtered and concentrated. The residue was purified by silica gelcolumn chromatography (10-15% EtOAc/n-heptanes) yielding4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylpiperidin-1-yl)-3-nitropyridine(40%). LC/MS=482.4 (M+H), R_(t)=1.26 min.

Synthesis of4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylpiperidin-1-yl)pyridin-3-amine

To a solution of4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylpiperidin-1-yl)-3-nitropyridine(1.0 equiv.) in ethanol, at a concentration of 0.05 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 14 hours. Atthis time the mixture was filtered through a pad of celite eluting withethanol. The volatiles were removed in vacuo yielding4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylpiperidin-1-yl)pyridin-3-amineLC/MS=452.4 (M+H), R_(t)=1.31 min.

Synthesis of (R)-tert-butyl4-((1R,2R)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-1-hydroxy-2-methyl-3-oxopropyl)-2,2-dimethyloxazolidine-3-carboxylate

To a solution of (R)-4-benzyl-3-propionyloxazolidin-2-one (1.0 equiv.)in DCM (0.13 M) was added TiCl₄ (1.0 equiv.) at −40° C. The mixture wasstirred at −40° C. for 10 min (yellow suspension), then DIPEA (2.5equiv.) was added (dark red solution) and stirred at 0° C. for 20 min.(R)-tert-butyl 4-formyl-2,2-dimethyloxazolidine-3-carboxylate (1.0equiv.) in DCM (0.5 M) was then added dropwise and the resulting mixturewas stirred for 1.5 hours. The reaction was quenched by the addition ofaqueous ammonium chloride and the mixture was extracted with ethylacetate. The organic phase was separated, washed with brine, dried withmagnesium sulfate, filtered, and concentrated. The residue was purifiedvia column chromatography eluting with ethyl acetate and hexanes (1:4)to give (R)-tert-butyl4-((1R,2R)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-1-hydroxy-2-methyl-3-oxopropyl)-2,2-dimethyloxazolidine-3-carboxylateas the major product (5:2) in 58% yield. LC/MS=363.3 (M+H-Boc), Rt=1.09min.

Synthesis of (R)-tert-butyl4-((1R,2R)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-1-(tert-butyldimethylsilyloxy)-2-methyl-3-oxopropyl)-2,2-dimethyloxazolidine-3-carboxylate

To a solution of (R)-tert-butyl4-((1R,2R)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-1-hydroxy-2-methyl-3-oxopropyl)-2,2-dimethyloxazolidine-3-carboxylate(1.0 equiv.) and lutidine (1.8 equiv.) in DCM (0.1M) was added TBSOTf(1.4 equiv.) at −40° C. The reaction mixture was stirred at −40° C. for2 hours. The solution was diluted with ethyl acetate and washed withsat. NaHCO₃, sat. NaCl, dried with magnesium sulfate, filtered, andconcentrated. The residue was purified by silica gel columnchromatography eluting with ethyl acetate and hexanes (1:4) to give(R)-tert-butyl4-((1R,2R)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-1-(tert-butyldimethylsilyloxy)-2-methyl-3-oxopropyl)-2,2-dimethyloxazolidine-3-carboxylateas the major product (5:2) in 83% yield. LC/MS=577.3 (M+H), Rt=1.33 min(Frac 65%-95% method).

Synthesis of (R)-tert-butyl4-((1R,2S)-1-(tert-butyldimethylsilyloxy)-3-hydroxy-2-methylpropyl)-2,2-dimethyloxazolidine-3-carboxylate

To a solution of (R)-tert-butyl4-((1R,2R)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-1-(tert-butyldimethylsilyloxy)-2-methyl-3-oxopropyl)-2,2-dimethyloxazolidine-3-carboxylate(1.0 equiv.) and ethanol (3.0 equiv.) in THF (0.09M) was added LiBH₄(3.0 equiv.) at −30° C. The reaction mixture was allowed to warm up to0° C. and stirred at that temperature for 3 hours. The solution was thendiluted with diethyl ether and 1N NaOH was added. The resulting mixturewas extracted with ethyl acetate, the organic layer was separated,washed with sat. NaCl, dried over magnesium sulfate, filtered, andconcentrated. The residue was purified via silica gel columnchromatography eluting with ethyl acetate and hexanes (1:4) to give(R)-tert-butyl4-((1R,2S)-1-(tert-butyldimethylsilyloxy)-3-hydroxy-2-methylpropyl)-2,2-dimethyloxazolidine-3-carboxylateas the major product (5:2 ratio) in 71% yield. LC/MS=304.3 (M+H-Boc),Rt=0.95 min (Frac 65%-95% method).

Synthesis of (R)-tert-butyl4-((1R,2S)-3-azido-1-(tert-butyldimethylsilyloxy)-2-methylpropyl)-2,2-dimethyloxazolidine-3-carboxylate

To a solution of (R)-tert-butyl4-((1R,2S)-1-(tert-butyldimethyl-silyloxy)-3-hydroxy-2-methylpropyl)-2,2-dimethyloxazolidine-3-carboxylate(1.0 equiv.), DIAD (2.0 equiv.), and PPh₃ (2.0 equiv.) in THF (0.18 M)was added DPPA (2.0 equiv., 1M solution in THF). The reaction mixturewas stirred at room temperature overnight. Upon removal of the volatilesunder vacuo, the residue was purified by silica gel columnchromatography eluting with ethyl acetate and hexanes (1:6) to give(R)-tert-butyl4-((1R,2S)-3-azido-1-(tert-butyldimethylsilyloxy)-2-methylpropyl)-2,2-dimethyloxazolidine-3-carboxylateas the major product (5:2) in 86% yield. LC/MS=329.3 (M+H-Boc), Rt=1.40min (Frac 65%-95% method).

Synthesis of tert-butyl(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-1-hydroxy-4-methylpentan-2-ylcarbamate

To a solution of (R)-tert-butyl4-((1R,2S)-3-azido-1-(tert-butyldimethylsilyloxy)-2-methylpropyl)-2,2-dimethyloxazolidine-3-carboxylate(1.0 equiv.) in EtOH (0.1 M) was added PPTS (1.3 equiv.) and the mixturewas refluxed for 2 days. The volatiles were removed under vacuo, theresidue was dissolved in DCM (0.1 M) and DIEA (1.5 equiv.) and Boc₂O(1.0 equiv.) were added to the reaction mixture. The solution wasstirred for 3 hours at room temperature. The solvents were removed underreduced pressure and the residue was diluted with ethyl acetate, washedwith water, aqueous NaHSO₄, aqueous NaHCO₃, sat. NaCl, the organic phasewas dried with magnesium sulfate, filtered, and concentrated. Theresidue was purified via silica gel column chromatography eluting withethyl acetate and hexanes (1:3) to give tert-butyl(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-1-hydroxy-4-methylpentan-2-ylcarbamateas the major isomer (5:2) in 70% yield. LC/MS=289.3 (M+H-Boc), Rt=0.76min (Frac 65%-95% method).

Synthesis of(2R,3R,4S)-5-azido-2-(tert-butoxycarbonylamino)-3-(tert-butyldimethylsilyloxy)-4-methylpentylmethanesulfonate

To a solution of tert-butyl(2R,3R,4S)-5-azido-3-(tert-butyldimethylsilyloxy)-1-hydroxy-4-methylpentan-2-ylcarbamate(1.0 equiv.) in pyridine (0.2 M) was added MsCl (1.3 equiv.) followed byDMAP (catalytic amount) at 0° C. The mixture was stirred at thattemperature for 1 hour. The solution was diluted with ether and ethylacetate (4:1), washed with aq. NaHSO₄, sat. NaHCO₃, brine, dried overmagnesium sulfate, filtered, and concentrated. The residue was purifiedby silica gel column chromatography eluting with ethyl acetate andhexanes (1:3) to give(2R,3R,4S)-5-azido-2-(tert-butoxycarbonylamino)-3-(tert-butyldimethylsilyloxy)-4-methylpentylmethanesulfonate as the major isomer (5:2) in 90% yield. LC/MS=367.3(M+H-Boc), Rt=0.81 min (Frac 65%-95% method).

Synthesis of tert-butyl(3R,4R,5S)-4-(tert-butyldimethylsilyloxy)-5-methylpiperidin-3-ylcarbamate

A solution of(2R,3R,4S)-5-azido-2-(tert-butoxycarbonylamino)-3-(tert-butyldimethylsilyloxy)-4-methylpentylmethanesulfonate in MeOH (0.09 M) was degassed with nitrogen for 20 min.DIEA (2.5 equiv.) was added, followed by 10% Pd/C (0.1 equiv.). Thereaction mixture was stirred under a hydrogen balloon for 2 hours. Thesolution was filtered and the filtrate was concentrated under vacuo toafford tert-butyl(3R,4R,5S)-4-(tert-butyldimethylsilyloxy)-5-methylpiperidin-3-ylcarbamateas the major isomer (5:2) in >99% yield. LC/MS=345.2 (M+H-Boc), Rt=0.95and 0.99 min.

Synthesis of tert-butyl(3R,4R,5S)-4-(tert-butyldimethylsilyloxy)-5-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate

To a solution of tert-butyl(3R,4R,5S)-4-(tert-butyldimethylsilyloxy)-5-methylpiperidin-3-ylcarbamate(1.0 equiv.) in i-PrOH (0.09 M) was added DIEA (2.5 equiv.) and4-chloro-3-nitropyridine (1.5 equiv.). The reaction mixture was stirredat 60° C. for 2 hours. The volatiles were removed under vacuo, theresidue was diluted with ethyl acetate and washed with sat. NaCl. Theorganic phase was dried with magnesium sulfate, filtered, andconcentrated. The crude material was purified by silica gel columnchromatography eluting with ethyl acetate and hexanes (1:2) to givetert-butyl(3R,4R,5S)-4-(tert-butyldimethylsilyloxy)-5-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamatein 76% yield. LC/MS=467.3 (M+H), Rt=1.09 min.

Synthesis of tert-butyl(3R,4R,5S)-1-(3-aminopyridin-4-yl)-4-(tert-butyldimethylsilyloxy)-5-methylpiperidin-3-ylcarbamate

A solution of tert-butyl(3R,4R,5S)-4-(tert-butyldimethylsilyloxy)-5-methyl-1-(3-nitropyridin-4-yl)piperidin-3-ylcarbamate(1.0 equiv.) in MeOH (0.05 M) was degassed with nitrogen for 20 min. 10%Pd/C (0.2 equiv.) was added to the mixture and the solution was stirredunder a hydrogen balloon for 3 hours. The reaction was filtered and thefiltrate was concentrated under reduced pressure to give tert-butyl(3R,4R,5S)-1-(3-aminopyridin-4-yl)-4-(tert-butyldimethylsilyloxy)-5-methylpiperidin-3-ylcarbamateas the desired product in 94% yield. LC/MS=437.4 (M+H), Rt=1.08 min.¹H-NMR (300 MHz, CDCl₃): δ 8.01 (s, 1H), 7.95 (d, J=6.0 Hz, 1H), 6.76(d, J=6.0 Hz, 1H), 4.44 (br s, 1H), 3.74 (br s, 2H), 3.59-3.55 (m, 1H),3.25-3.13 (m, 2H), 2.47-2.35 (m, 2H), 1.89 (br s, 2H), 1.44 (s, 9H),1.04 (d, J=6.0, 3H), 0.92 (s, 9H), 0.13 (d, J=9.0, 6H).

Synthesis of tert-butyl(2R)-1-(benzyloxy)-3-hydroxy-4-methylhex-5-en-2-ylcarbamate

To a solution of N-Boc, O-benzyl-D-Serine aldehyde (1.0 equiv) in DCM(0.1M) at −78° C. under an Ar atmosphere was added(Z)-2-(but-2-enyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.1 equiv)and the clear solution stirred for 16 hours as it warmed to rt. Thesolution was added to EtOAc and was washed with H₂O (3×), andNaCl_((sat.)), dried over MgSO₄ and purified by silica gelchromatography (15% EtOAc/hexanes) to yield tert-butyl(2R)-1-(benzyloxy)-3-hydroxy-4-methylhex-5-en-2-ylcarbamate (54%) as a3:1 mixture of isomers as judged by ¹H NMR. LCMS (m/z): 236.3 (MH⁺-Boc);LC R_(t)=4.37 and 4.51 min.

Synthesis of (4R)-4-(benzyloxymethyl)-5-(but-3-en-2-yl)oxazolidin-2-one

To a solution of (2R)-1-(benzyloxy)-3-hydroxy-4-methylhex-5-en-2-in THF(0.1 M) was added 60% sodium hydride in mineral oil (1.5 equiv.). Afterstirring for 3 days, the reaction was quenched by addition ofNH₄Cl_((sat.)) and solution was diluted with EtOAc and washed withNH₄Cl_((sat.)) and NaCl_((sat.)), dried over MgSO₄ and purified bysilica gel chromatography (50% EtOAc/hexanes) to yield(4R)-4-(benzyloxymethyl)-5-(but-3-en-2-yl)oxazolidin-2-one (89%) as a3:1 mixture. LCMS (m/z): 262.2 (MH⁺); LC R_(t)=3.47 min.

Synthesis of(4R)-4-(benzyloxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one

To a solution of(4R)-4-(benzyloxymethyl)-5-(but-3-en-2-yl)oxazolidin-2-one (1.0 equiv.)in 2:1 MeOH/H₂O (0.04 M) was added osmium tetroxide 4% in H₂O (0.07equiv) and sodium periodate (3.0 equiv.). After stirring for 3 hours,the white precipitate was filtered and rinsed with EtOAc. The combinedfiltrate was concentrated in vacuo and the residue was dissolved inEtOAc, washed with NaCl_((sat.)), dried over MgSO₄, filtered andconcentrated. The crude aldehyde was dissolved in EtOH (0.08 M) and uponcooling to 0° C., sodium borohydride (2.0 equiv.) was added. Afterstirring for 15 hours and coming to room temperature the reaction wasquenched by addition of H₂O. After stirring for 20 minutes, the EtOH wasremoved in vacuo, EtOAc was added and the solution was washed with 1NHCl, NaHCO_(3(sat.)) and NaCl_((sat.)), dried over MgSO₄, filtered andconcentrated yielding after purification by silica gel chromatography(4R)-4-(benzyloxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one as a3:1 mixture of isomers (60%). LCMS (m/z): 266.1 (MH⁺); LC R_(t)=2.28min.

Synthesis of(4R)-4-(hydroxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one

To a solution of(4R)-4-(benzyloxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one (1.0equiv.) in methanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 15 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding(4R)-4-(hydroxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one (99%).LCMS (m/z): 176.1 (MH⁺).

Synthesis of 2-((4R)-2-oxo-4-(tosyloxymethyl)oxazolidin-5-yl)-propyl4-methylbenzenesulfonate

To a solution of(4R)-4-(hydroxymethyl)-5-(1-hydroxypropan-2-yl)oxazolidin-2-one (1.0equiv.) in pyridine (0.15 M) at 0° C. was addedp-toluenesulfonylchloride (2.1 equiv.). The solution was allowed to warmto rt as it stirred for 14 hours, at which time EtOAc was added and thesolution was washed with H₂O (3×), CuSO_(4(sat.)) (2×), H₂O,Na₂CO_(3(sat.)) and NaCl_((sat.)), dried over MgSO₄, filtered,concentrated and purified by silica gel chromatography (75%EtOAc/hexanes eluent) yielding2-((4R)-2-oxo-4-(tosyloxymethyl)oxazolidin-5-yl)propyl4-methylbenzenesulfonate (68%). LCMS (m/z): 484.1 (MH⁺); LC R_(t)=4.06min.

Synthesis of (3aR,7R,7aS)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-oneand(3aR,7S,7aR)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one

A solution of 2-((4R)-2-oxo-4-(tosyloxymethyl)oxazolidin-5-yl)propyl4-methylbenzenesulfonate (1.0 equiv.), diisopropylethyl amine (3.0equiv.) and para-methoxybenzylamine (1.5 equiv.) in NMP (0.05 M) washeated at 100° C. for 14 hours. The solution was purified directly by RPHPLC. The product fractions were desalted by addition to EtOAc andNa₂CO_(3(s)), washed further with NaCl(sat.), dried over MgSO₄ andconcentrated yielding two separate isomers of(3aR,7R,7aS)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-oneand(3aR,7S,7aR)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one(27% and 8%). LCMS (m/z): 277.2 (MH⁺) at 0.40 and 0.42 min.

Synthesis of(3aR,7R,7aS)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one

To a solution of(3aR,7R,7aS)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one(1.0 equiv.) in methanol, at a concentration of 0.1M, was added 20%palladium hydroxide on carbon (0.3 eq.). The resultant heterogeneoussolution was put under an atmosphere of hydrogen and was stirred for 2hours. At this time the mixture was filtered through a pad of celiteeluting with methanol. The volatiles were removed in vacuo yielding(3aR,7R,7aS)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (99%).LCMS (m/z): 157.1 (MH⁺) at 0.16 min.

Synthesis of (3aR,7R,7aS)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

A solution of 4-chloro-3-nitropyridine (1.3 equiv.) and(3aR,7R,7aS)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (1.5equiv.) in CH₂Cl₂, at a concentration of 0.1 M, was stirred at rt for 48hours at which piperidine (0.4 equiv) was added to consume excess4-chloro-3-nitropyridine. After stirring for an additional 2 hours,di-tert-butyl dicarbonate (2.0 equiv.) and dimethylaminopyridine (0.1equiv.) were added. After stirring for 4 hours, the solution waspartitioned between EtOAc and NaHCO_(3(sat.)), was washed further withNaHCO_(3(sat.)), and NaCl_((sat.)), was dried over MgSO₄, was filteredand purified by silica gel chromatography yielding(3aR,7R,7aS)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)carboxylate(62%). LCMS (m/z): 379.0 (MH⁺) at 0.58 min.

Synthesis of (3aR,7R,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

To a solution of (3aR,7R,7aS)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 14 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding(3aR,7R,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate. LCMS (m/z): 349.1 (MH⁺); LC R_(t)=2.06 min.

Synthesis of(3aR,7S,7aR)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one

To a solution of(3aR,7S,7aR)-5-(4-methoxybenzyl)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 20%palladium hydroxide on carbon (0.3 eq.). The resultant heterogeneoussolution was put under an atmosphere of hydrogen and was stirred for 2hours. At this time the mixture was filtered through a pad of celiteeluting with methanol. The volatiles were removed in vacuo yielding(3aR,7S,7aR)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (99%).LCMS (m/z): 157.1 (MH⁺) at 0.17 min.

Synthesis of (3aR,7S,7aR)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

A solution of 4-chloro-3-nitropyridine (1.3 equiv.) and(3aR,7S,7aR)-7-methylhexahydrooxazolo[4,5-c]pyridin-2(3H)-one (1.5equiv.) in CH₂Cl₂, at a concentration of 0.1 M, was stirred at rt for 48hours at which piperidine (0.4 equiv) was added to consume excess4-chloro-3-nitropyridine. After stirring for an additional 2 hours,di-tert-butyl dicarbonate (2.0 equiv.) and dimethylaminopyridine (0.1equiv.) were added. After stirring for 4 hours, the solution waspartitioned between EtOAc and NaHCO_(3(sat.)), was washed further withNaHCO_(3(sat.)), and NaCl_((sat.)), was dried over MgSO₄, was filteredand purified by silica gel chromatography (75% EtOAc/hexanes eluent)yielding (3aR,7S,7aR)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate(35%). LCMS (m/z): 379.0 (MH⁺). LC R_(t)=2.42 min.

Synthesis of (3aR,7R,7aS)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate

To a solution of (3aR,7S,7aR)-tert-butyl7-methyl-5-(3-nitropyridin-4-yl)-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate(1.0 equiv.) in methanol, at a concentration of 0.1 M, was added 10%palladium on carbon (0.1 eq.). The resultant heterogeneous solution wasput under an atmosphere of hydrogen and was stirred for 14 hours. Atthis time the mixture was filtered through a pad of celite eluting withmethanol. The volatiles were removed in vacuo yielding(3aR,7S,7aR)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrooxazolo[4,5-c]pyridine-3(2H)-carboxylate.LCMS (m/z): 349.1 (MH⁺); LC R_(t)=2.18 min.

Method 1

Synthesis of methyl 3-amino-6-(2,6-difluorophenyl)picolinate

A solution of methyl 3-amino-6-bromopicolinate (1.0 equiv.),2,6-difluorophenyl-boronic acid (3.0 equiv), and Pd(dppf)Cl₂-DCM (0.1equiv.) in 3:1 DME/2M Na₂CO₃ (0.5 M) was subjected to microwaveirradiation at 120° C. for 15 min intervals. The reaction was filteredand washed with EtOAc. The organic was partitioned with H₂O (25 mL), wasfurther washed with NaCl_((sat.)) (25 mL), was dried over MgSO₄, and thevolatiles were removed in vacuo. The residue was diluted in EtOAc andpassed through a silica gel plug and the volatiles were removed in vacuoyielding methyl 3-amino-6-(2,6-difluorophenyl)picolinate (47%). LCMS(m/z): 265.1 (MH⁺); LC R_(t)=2.70 min

Synthesis of 6-(2,3-difluorophenyl)-5-fluoropicolinic acid

To a solution of 6-bromo-5-fluoropicolinic acid (1.0 equiv.) in DME and2M Na₂CO₃ (3:1, 0.25 M) was added 2,3-difluorophenylboronic acid (1.3equiv.) and Pd(dppf)Cl₂-DCM (0.05 equiv.) in a microwave vial. The vialwas heated in the microwave at 120° C. for 30 minutes. The mixture wasdiluted with ethyl acetate and 1N NaOH was added. The organic phase wasseparated and extracted three more times with 1N NaOH and once with 6NNaOH. The combined aqueous phases were filtered and acidified to pH 1 bythe addition of concentrated HCl and extracted with ethyl acetate. Theorganic layer was dried over magnesium sulfate, filtered, andconcentrated to give 6-(2,3-difluorophenyl)-5-fluoropicolinic acid in78%. LC/MS=254.1 (M+H), Rt=0.75 min.

Method 2

Synthesis of 3-amino-6-(2,6-difluorophenyl)picolinic acid

To a solution of methyl 3-amino-6-(2,6-difluorophenyl)picolinate (1.0equiv) in THF (0.5 M), was added 1M LiOH (4.0 equiv). After stirring for4 hours at 60° C., 1N HCl (4.0 equiv.) was added and the THF was removedin vacuo. The resulting solid was filtered and rinsed with cold H₂O(3×20 mL) to yield 3-amino-6-(2,6-difluorophenyl)picolinic acid (90%).LCMS (m/z): 251.1 (MH⁺); LC R_(t)=2.1 min.

Synthesis of 3-amino-6-(2-fluoro-5-propoxyphenyl)picolinic acid

Method 1 was followed using 3-amino-6-bromopicolinic acid (1.0 equiv.)and 2-fluoro-5-propoxyphenylboronic acid (1.5 equiv.) andPd(dppf)Cl₂-DCM (0.05 equiv.) to give3-amino-6-(2-fluoro-5-propoxyphenyl)picolinic acid in 75% yield.LC/MS=291.0 (M+H), Rt=0.81 min.

Synthesis of 3-amino-5-fluoro-6-(2-fluoro-5-propoxyphenyl)picolinic acid

Method 1 was followed using 3-amino-6-bromo-5-fluoropicolinic acid (1.0equiv.) and 2-fluoro-5-propoxyphenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.05 equiv.) to give3-amino-5-fluoro-6-(2-fluoro-5-propoxyphenyl)picolinic acid in 28%yield. LC/MS=309.1 (M+H), Rt=1.00 min.

Synthesis of methyl 3-amino-5-fluoro-6-(2-fluorophenyl)picolinate

Method 1 was followed using methyl 3-amino-6-bromo-5-fluoropicolinate(1.0 equiv.) and 2-fluoro-phenylboronic acid (1.5 equiv.) andPd(dppf)Cl₂-DCM (0.05 equiv.) to give methyl3-amino-5-fluoro-6-(2-fluorophenyl)picolinate in >99% yield. LC/MS=265.0(M+H), Rt=0.77 min.

Synthesis of 3-amino-5-fluoro-6-(2-fluorophenyl)picolinic acid

Method 2 was followed using3-amino-5-fluoro-6-(2-fluorophenyl)picolinate (1.0 equiv.) and LiOH (5.0equiv.) to give 3-amino-5-fluoro-6-(2-fluorophenyl)picolinic acid in 90%yield. LC/MS=251.1 (M+H), Rt=0.80 min.

Synthesis of methyl 3-amino-6-(2,6-difluorophenyl)-5-fluoropicolinate

Method 1 was followed using methyl 3-amino-6-bromo-5-fluoropicolinate(1.0 equiv.) and 2,6-difluorophenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.05 equiv.) to give3-amino-6-(2,6-difluorophenyl)-5-fluoropicolinate in 94% yield.LC/MS=283.0 (M+H), Rt=0.76 min.

Synthesis of 3-amino-6-(2,6-difluorophenyl)-5-fluoropicolinic acid

Method 2 was followed using3-amino-6-(2,6-difluorophenyl)-5-fluoropicolinate (1.0 equiv.) and LiOH(1.0 equiv.) to give 3-amino-6-(2,6-difluorophenyl)-5-fluoropicolinicacid in 79% yield. LC/MS=269.0 (M+H), Rt=0.79 min.

Synthesis of 5-fluoro-6-(2-fluorophenyl)picolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and 2-fluorophenylboronic acid (1.3 equiv.) and Pd(dppf)Cl₂-DCM (0.05equiv.) to give 5-fluoro-6-(2-fluorophenyl)picolinic acid in 43% yield.LC/MS=236.1 (M+H), Rt=0.72 min.

Synthesis of 6-(3,4-difluorophenyl)-5-fluoropicolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and 3,4-difluorophenylboronic acid (1.3 equiv.) and Pd(dppf)Cl₂-DCM(0.05 equiv.) to give 6-(3,4-difluorophenyl)-5-fluoropicolinic acid in70% yield. LC/MS=254.1 (M+H), Rt=0.81 min.

Synthesis of 6-(2,5-difluorophenyl)-5-fluoropicolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and 2,5-difluorophenylboronic acid (1.3 equiv.) and Pd(dppf)Cl₂-DCM(0.05 equiv.) to give 6-(2,5-difluorophenyl)-5-fluoropicolinic acid in80% yield. LC/MS=254.1 (M+H), Rt=0.74 min.

Synthesis of 6-(2,4-difluorophenyl)-5-fluoropicolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and 2,4-difluorophenylboronic acid (1.3 equiv.) and Pd(dppf)Cl₂-DCM(0.05 equiv.) to give 6-(2,4-difluorophenyl)-5-fluoropicolinic acid in79% yield. LC/MS=254.1 (M+H), Rt=0.75 min.

Synthesis of 5-fluoro-6-(2-fluoro-5-propoxyphenyl)picolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and 2-fluoro-5-propoxyphenylboronic acid (1.5 equiv.) andPd(dppf)Cl₂-DCM (0.05 equiv.) to give5-fluoro-6-(2-fluoro-5-propoxyphenyl)picolinic acid. LC/MS=294.2 (M+H),Rt=0.95 min.

Synthesis of 6-(2-fluorophenyl)picolinic acid

Method 1 was followed using 6-bromopicolinic acid (1.0 equiv.) and2-fluorophenylboronic acid (1.5 equiv.) and Pd(dppf)Cl₂-DCM (0.05equiv.) to give 6-(2-fluorophenyl)picolinic acid in 93% yield.LC/MS=218.0 (M+H), Rt=0.66 min.

Synthesis of 6-(2,6-difluorophenyl)picolinic acid

Method 1 was followed using 6-bromopicolinic acid (1.0 equiv.) and2,6-difluorophenylboronic acid (1.5 equiv.) and Pd(dppf)Cl₂-DCM (0.05equiv.) to give 6-(2,6-difluorophenyl)picolinic acid in 38% yield.LC/MS=236.0 (M+H), Rt=0.87 min.

Synthesis of 6-(2-fluoro-5-methoxyphenyl)picolinic acid

Method 1 was followed using 6-bromopicolinic acid (1.0 equiv.) and2-fluoro-5-methoxyphenylboronic acid (1.3 equiv.) and Pd(dppf)Cl₂-DCM(0.15 equiv.) to give 6-(2-fluoro-5-methoxyphenyl)picolinic acid in 95%yield. LC/MS=248.2 (M+H), Rt=0.78 min.

Synthesis of 6-(2-fluoro-5-propoxyphenyl)picolinic acid

Method 1 was followed using 6-bromopicolinic acid (1.0 equiv.) and2-fluoro-5-propoxyphenylboronic acid (1.5 equiv.) and Pd(dppf)Cl₂-DCM(0.15 equiv.) to give 6-(2-fluoro-5-propoxyphenyl)picolinic acid in 20%yield. LC/MS=276.0 (M+H), Rt=0.87 min.

Synthesis of 6-(2,6-difluoro-4-methoxyphenyl)picolinic acid

Method 1 was followed using 6-bromopicolinic acid (1.0 equiv.) and2,6-difluoro-4-methoxyphenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.15 equiv.) to give6-(2,6-difluoro-4-methoxyphenyl)picolinic acid in 42% yield. LC/MS=266.1(M+H), Rt=0.75 min.

Synthesis of 3-fluoro-6-(2-fluorophenyl)picolinic acid

Method 1 was followed using 6-bromo-3-fluoropicolinic acid (1.0 equiv.)and 2-fluorophenylboronic acid (1.5 equiv.) and Pd(dppf)Cl₂-DCM (0.05equiv.) to give 3-fluoro-6-(2-fluorophenyl)picolinic acid in 81% yield.LC/MS=236.1 (M+H), Rt=0.72 min.

Synthesis of 3-fluoro-6-(2-fluoro-5-methoxyphenyl)picolinic acid

Method 1 was followed using 6-bromo-3-fluoropicolinic acid (1.0 equiv.)and 2-fluoro-5-methoxyphenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.15 equiv.) to give3-fluoro-6-(2-fluoro-5-methoxyphenyl)picolinic acid in 89% yield.LC/MS=266.1 (M+H), Rt=0.79 min.

Synthesis of 5-fluoro-6-(2-fluoro-5-methoxyphenyl)picolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and 2-fluoro-5-methoxyphenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.15 equiv.) to give5-fluoro-6-(2-fluoro-5-methoxyphenyl)picolinic acid in 86% yield.LC/MS=266.1 (M+H), Rt=0.79 min.

Synthesis of 6-(4-(benzyloxy)-2-fluorophenyl)-5-fluoropicolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and 4-(benzyloxy)-2-fluorophenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.15 equiv.) to give6-(4-(benzyloxy)-2-fluorophenyl)-5-fluoropicolinic acid in 28% yield.LC/MS=342.1 (M+H), Rt=1.05 min.

Synthesis of 6-(4-(benzyloxy)-2-fluorophenyl)-3-fluoropicolinic acid

Method 1 was followed using 6-bromo-3-fluoropicolinic acid (1.0 equiv.)and 4-(benzyloxy)-2-fluorophenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.15 equiv.) to give6-(4-(benzyloxy)-2-fluorophenyl)-3-fluoropicolinic acid in 41% yield.LC/MS=342.1 (M+H), Rt=1.06 min.

Synthesis of 6-(2,6-difluoro-4-methoxyphenyl)-3-fluoropicolinic acid

Method 1 was followed using 6-bromo-3-fluoropicolinic acid (1.0 equiv.)and 2,6-difluoro-4-methoxyphenylboronic acid (1.3 equiv.) andPd(dppf)Cl₂-DCM (0.15 equiv.) to give6-(2,6-difluoro-4-methoxyphenyl)-3-fluoropicolinic acid in 9% yield.LC/MS=284.0 (M+H), Rt=0.74 min.

Synthesis of 6-cyclohexenyl-5-fluoropicolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and cyclohexenylboronic acid (1.3 equiv.) and Pd(dppf)Cl₂-DCM (0.15equiv.) to give 6-cyclohexenyl-5-fluoropicolinic acid in 61% yield.LC/MS=222.0 (M+H), Rt=0.52 min.

Method 3

Synthesis of 6-cyclohexyl-5-fluoropicolinic acid

To a degassed solution of 6-cyclohexenyl-5-fluoropicolinic acid (1.0equiv.) in MeOH (0.07M) was added 10% Pd/C (0.1 equiv.) and the reactionwas stirred under a hydrogen balloon overnight. The solution was thenfiltered, rinsed with MeOH, and the filtrate was concentrated to afford6-cyclohexyl-5-fluoropicolinic acid in 65% yield. LC/MS=224.2 (M+H),Rt=0.95 min.

Synthesis of 5-fluoro-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)picolinic acid

Method 1 was followed using 6-bromo-5-fluoropicolinic acid (1.0 equiv.)and4,4,5,5-tetramethyl-2-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1,3,2-dioxaborolane(2.0 equiv.) and Pd(dppf)Cl₂-DCM (0.2 equiv.) to give5-fluoro-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)picolinic acid. LC/MS=280.2(M+H), Rt=0.66 min.

Synthesis of methyl5-fluoro-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)picolinate

To a solution of 5-fluoro-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)picolinicacid (1.0 equiv.) in DCM (0.3 M) was added EDC-HCl (1.0 equiv.), DMAP(1.0 equiv.), and MeOH (10 equiv.). The reaction mixture was allowed tostir at room temperature for 5 days, then diluted with ethyl acetate,washed with water, brine, dried over magnesium sulfate, filtered andconcentrated. The crude product was purified by silica gel columnchromatography eluting with 25-50% ethyl acetate in hexanes to yieldmethyl 5-fluoro-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)picolinate as thedesired product in 35% yield. LC/MS=294.2 (M+H), Rt=0.79 min.

Synthesis of methyl 5-fluoro-6-(1,4-dioxaspiro[4.5]decan-8-yl)picolinate

To a degassed solution of methyl5-fluoro-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)picolinate (1.0 equiv.) inMeOH (0.07M) was added 10% Pd/C (0.1 equiv.) and the reaction wasstirred under a hydrogen balloon overnight. The solution was thenfiltered, rinsed with MeOH, and the filtrate was concentrated to affordmethyl 5-fluoro-6-(1,4-dioxaspiro[4.5]decan-8-yl)picolinate in 91%yield. LC/MS=296.2 (M+H), Rt=0.83 min.

Synthesis of methyl 5-fluoro-6-(4-oxocyclohexyl)picolinate

To a solution of methyl5-fluoro-6-(1,4-dioxaspiro[4.5]decan-8-yl)picolinat (1.0 equiv.) inacetone and water (1:1, 0.04 M) was added oxalic acid dehydrate (2.0equiv.) and the reaction mixture was stirred for 3 days. The solutionwas then neutralized by the addition of solid NaHCO₃, the mixture wasadded to ethyl acetate and brine, the organic phase was dried overmagnesium sulfate, filtered, and concentrated. Methyl5-fluoro-6-(4-oxocyclohexyl)picolinate was obtained in 98% yield.LC/MS=252.1 (M+H), Rt=0.68 min.

Synthesis of methyl 5-fluoro-6-(4-hydroxycyclohexyl)picolinate

To a 0° C. solution of methyl 5-fluoro-6-(4-oxocyclohexyl)picolinate(1.0 equiv.) win MeOH (0.08 M) was added NaBH₄. The solution was allowedto warm to room temperature overnight then partitioned between ethylacetate and brine, the organic phase was dried over magnesium sulfate,filtered, and concentrated to give methyl5-fluoro-6-(4-hydroxycyclohexyl)picolinate as a mixture of two isomers(5:1). LC/MS=254.2 (M+H), Rt=0.63 min.

Synthesis of methyl6-(4-(tert-butyldimethylsilyloxy)cyclohexyl)-5-fluoropicolinate

To a solution of methyl 5-fluoro-6-(4-hydroxycyclohexyl)picolinate (1.0equiv.) in DMF (0.15 M) was added imidazole (4.0 equiv.) and TBDMSC1(2.5 equiv.). The reaction mixture was stirred at room temperature for 2days, then added to ethyl acetate, washed with water, brine, dried overmagnesium sulfate, filtered and concentrated to give methyl6-(4-(tert-butyldimethylsilyloxy)cyclohexyl)-5-fluoropicolinate in 97%yield as a mixture of isomers (3:1). LC/MS=368.3 (M+H), Rt=1.4 and 1.42min.

Synthesis of6-(4-(tert-butyldimethylsilyloxy)cyclohexyl)-5-fluoropicolinic acid

To a solution of methyl6-(4-(tert-butyldimethylsilyloxy)cyclohexyl)-5-fluoropicolinate (1.0equiv.) in THF/MeOH (2:1, 0.09 M) was added LiOH (1.5 equiv.). Thereaction mixture was stirred overnight at room temperature, then 1N HCland ethyl acetate were added, the organic phase was washed with brine,dried over magnesium sulfate, filtered and concentrated to give6-(4-(tert-butyldimethylsilyloxy)cyclohexyl)-5-fluoropicolinic acid as amixture of isomers (3:1) in 82% yield. LC/MS=354.2 (M+H), Rt=1.38 and1.41 min.

Synthesis of 6-bromo-5-fluoropicolinic acid

To 2-bromo-3-fluoro-6-methylpyridine (1.0 equiv.) in H₂O (30 mL) wasadded potassium permanganate (1.0 equiv.). The solution was heated at100° C. for 5 hours at which time more potassium permanganate (1.0equiv.) was added. After heating for an additional 48 hours the materialwas filtered through celite (4 cm×2 inches) and rinsed with H₂O (150mL). The combined aqueous was acidified with 1N HCl to pH=4, extractedwith ethyl acetate (200 mL), washed with NaCl(sat.), dried over MgSO₄,filtered and concentrated to yield 6-bromo-5-fluoropicolinic acid (17%)as a white solid. LCMS (m/z): 221.9 (MH⁺); LC Rt=2.05 min.

Method 4

Synthesis of 2-(2,6-difluorophenyl)-3-fluoro-6-methylpyridine

To a solution of 2-bromo-3-fluoro-6-methylpyridine (1.0 equiv.) in THFand Water (10:1, 0.2 M) was added 2,6-difluorophenylboronic acid (2.0equiv.) and potassium fluoride (3.3 equiv.). The reaction was degassedfor 10 minutes, then Pd₂(dba)₃ (0.05 equiv.) was added, followed bytri-t-butylphosphine (0.1 equiv.). The reaction was stirred to 60° C.for 1 hour at which point, all starting material was consumed asindicated by LC/MS. The reaction was allowed to cool to roomtemperature, partitioned with ethyl acetate and water, the organic phasewas dried with sodium sulfate, filtered, and concentrated. The crudematerial was diluted in EtOH to 0.1 M, and 0.5 equiv. of NaBH₄ was addedto reduce the dba. The reaction was stirred for one hour at roomtemperature, then quenched with water and concentrated under vacuo toremove the ethanol. The product was extracted in ether, washed withbrine, the organics were dried over sodium sulfate, filtered, andconcentrated. The crude material was loaded on silica gel and purifiedvia column chromatography (ISCO) eluting with hexanes and ethyl acetate(0%-10% ethyl acetate). The pure fractions were combined, andconcentrated to yield 2-(2,6-difluorophenyl)-3-fluoro-6-methylpyridineas a light yellow oil in 86% yield. LC/MS=224.0 (M+H), Rt=0.84 min.

Method 5

Synthesis of 6-(2,6-difluorophenyl)-5-fluoropicolinic acid

To a solution of 2-(2,6-difluorophenyl)-3-fluoro-6-methylpyridine (1.0equiv.) in water (0.05 M) was added KMnO₄ (2.0 equiv.) and the reactionwas heated to reflux overnight. Another 2.0 equiv. of KMnO₄ were addedand stirred at reflux for another 8 hours. The solution was cooled toroom temperature, filtered through Celite and washed with water. Thefiltrate was acidified with 6N HCl to pH=3, the white precipitate wasfiltered. The filtrate was further acidified to pH=1 and filtered again.The filtrate was extracted with ethyl acetate until no more product inthe aqueous layer. The organic phase was washed with brine and driedover magnesium sulfate, filtered, and concentrated. The residue wasdissolved in ethyl acetate, washed with 1N NaOH, the aqueous layer wasacidified to pH=1 and the white crystals were filtered. The combinedproducts yielded 6-(2,6-difluorophenyl)-5-fluoropicolinic acid in 32%yield as a white solid. LC/MS=254.0 (M+H), Rt=0.71 min.

Synthesis of 6-(2,6-difluorophenyl)-3-fluoro-2-methylpyridine

To a solution of 6-bromo-3-fluoro-2-methylpyridine (1.0 equiv.) inethanol and toluene (1:1, 0.2 M) was added 2,6-difluorophenylboronicacid, DIEA (5 equiv.) and Pd(PPh₃)₄ (0.2 equiv.). The reaction washeated in the microwave at 120° C. for 30 min. The solution was filteredand rinsed with ethyl acetate. The volatiles were removed in vacuo andthe crude was purified via silica gel column chromatography eluting withethyl acetate and hexanes (2.5-20% ethyl acetate). Upon concentration ofthe pure fractions, 6-(2,6-difluorophenyl)-3-fluoro-2-methylpyridine wasisolated in 88% yield. LC/MS=224.1 (M+H), Rt=0.87 min.

Synthesis of 6-(2,6-difluorophenyl)-3-fluoropicolinic acid

Method 5 was followed using6-(2,6-difluorophenyl)-3-fluoro-2-methylpyridine (1.0 equiv.) andpotassium permanganate (6.0 equiv.) to give6-(2,6-difluorophenyl)-3-fluoropicolinic acid in 30% yield. LC/MS=254.1(M+H), Rt=0.70 min.

Synthesis of 2-(2,6-difluoro-3-methoxyphenyl)-3-fluoro-6-methylpyridine

Method 4 was followed using 2-bromo-3-fluoro-6-methylpyridine (1.0equiv.) and 2,6-difluoro-3-methoxyphenylboronic acid (2.0 equiv.) togive 2-(2,6-difluoro-3-methoxyphenyl)-3-fluoro-6-methylpyridine in 60%yield. LC/MS=254.1 (M+H), Rt=0.85 min.

Synthesis of 6-(2,6-difluoro-3-methoxyphenyl)-5-fluoropicolinic acid

Method 5 was followed using2-(2,6-difluoro-3-methoxyphenyl)-3-fluoro-6-methylpyridine (1.0 equiv.)and potassium permanganate (4.0 equiv.) to give6-(2,6-difluoro-3-methoxyphenyl)-5-fluoropicolinic acid in 27% yield.LC/MS=284.1 (M+H), Rt=0.75 min.

Synthesis of 3-fluoro-6-methyl-2-(2,3,5-trifluorophenyl)pyridine

To a solution of 2-bromo-3-fluoro-6-methylpyridine (1.0 equiv.) indioxane (0.2 M) was added 2,3,5-trifluorophenylboronic acid andPd(dppf)Cl₂-DCM (0.1 equiv.). Aqueous sodium carbonate (2M solution, 2.0equiv.) was added and the reaction was heated in the microwave at 120°C. for 15 min. The solution was partitioned between ethyl acetate andsat. NaHCO₃, the organic phase was washed with brine, dried withmagnesium sulfate, filtered, and concentrated. The crude material waspurified via silica gel column chromatography eluting with ethyl acetateand hexanes (1:3) to give3-fluoro-6-methyl-2-(2,3,5-trifluorophenyl)pyridine in 87% yield.LC/MS=242.1 (M+H), Rt=0.98 min.

Synthesis of 5-fluoro-6-(2,3,5-trifluorophenyl)picolinic acid

To a solution of 3-fluoro-6-methyl-2-(2,3,5-trifluorophenyl)pyridine(1.0 equiv.) in water and t-BuOH (2:1, 0.06 M) was added potassiumpermanganate (10 equiv.) and the solution was heated at 90° C. for 5hours. Upon cooling to room temperature, the solution was filtered, andthe filtrate was concentrated under reduced pressure to yield5-fluoro-6-(2,3,5-trifluorophenyl)picolinic acid in 89% yield.LC/MS=272.0 (M+H), Rt=0.80 min.

Synthesis of methyl 6-bromo-5-fluoropicolinate

To a solution of 6-bromo-5-fluoropicolinic acid (1.0 equiv.) in methanol(0.2 M) was added H₂SO₄ (4.2 equiv.) and the reaction was stirred atroom temperature for two hours. Upon completion of the reaction asmonitored by LC/MS, the reaction was diluted with ethyl acetate andquenched slowly with saturated aqueous NaHCO₃. The reaction was pouredinto a separatory funnel and extracted with ethyl acetate. The organicphase was dried with magnesium sulfate, filtered, and concentrated invacuo to provide methyl 6-bromo-5-fluoropicolinate as a white solid(>99%). LC/MS=233.9/235.9 (M+H), Rt=0.69 min.

Method 6

Synthesis of methyl6-(3-(benzyloxy)-2,6-difluorophenyl)-5-fluoropicolinate

To a solution of methyl 6-bromo-5-fluoropicolinate (1.0 equiv.) in THFand water (10:1, 0.1 M) was added3-(benzyloxy)-2,6-difluorophenylboronic acid (2.5 equiv.) and potassiumfluoride (3.3 equiv.). The reaction was degassed with nitrogen, thenPd₂(dba)₃ (0.25 equiv.) and tri-tert-butylphosphine (0.5 equiv.) wereadded and the reaction was heated to 80° C. for one hour. LC/MS analysisindicated complete conversion of the starting material to product. Thereaction was cooled to room temperature, then concentrated in vacuo andfused to silica gel. The crude product was purified by ISCO flashchromatography eluting with ethyl acetate and hexanes (0% to 30% ethylacetate) to provide methyl6-(3-(benzyloxy)-2,6-difluorophenyl)-5-fluoropicolinate as the desiredproduct as a light yellow oil in 96% yield. LC/MS=374.0 (M+H), Rt=1.07min.

Synthesis of methyl 6-(3-(benzyloxy)-2,6-difluorophenyl)picolinate

Method 6 was followed using methyl 6-bromopicolinate (1.0 equiv.) and3-(benzyloxy)-2,6-difluorophenylboronic acid (2.5 equiv.) to give methyl6-(3-(benzyloxy)-2,6-difluorophenyl)picolinate as a light yellow solidin 95% yield. LC/MS=356.2 (M+H), Rt=1.03 min.

Synthesis of methyl 6-(2,6-difluoro-4-methoxyphenyl)-5-fluoropicolinate

Method 6 was followed using methyl 6-bromopicolinate (1.0 equiv.) and2,6-difluoro-4-methoxyphenylboronic acid (2.5 equiv.) to give methyl6-(2,6-difluoro-4-methoxyphenyl)-5-fluoropicolinate as a white solid in85% yield. LC/MS=298.0 (M+H), Rt=0.89 min.

Synthesis of 6-(2,6-difluoro-4-methoxyphenyl)-5-fluoropicolinic acid

To a solution of methyl6-(2,6-difluoro-4-methoxyphenyl)-5-fluoropicolinate (1.0 equiv.) inTHF/MeOH (2:1, 0.09 M) was added LiOH (1.5 equiv.) and the reaction wasstirred at room temperature for 1 hour. The solution was quenched with1N HCl, extracted with ethyl acetate, washed with brine, dried withsodium sulfate, filtered and concentrated to give6-(2,6-difluoro-4-methoxyphenyl)-5-fluoropicolinic acid in 84% yield.LC/MS=284.1 (M+H), Rt=0.76 min.

Method 7

Synthesis of methyl 6-(2,6-difluoro-3-hydroxyphenyl)-5-fluoropicolinate

To a solution of methyl6-(3-(benzyloxy)-2,6-difluorophenyl)-5-fluoropicolinate (1.0 equiv.) inmethanol (0.1 M) was added 10% Pd/C (0.1 equiv.) in ethyl acetate. Thereaction was placed under an atmosphere of hydrogen and stirred for 2hours. Upon completion, the solution was filtered over a pad of Celite,the pad was washed with methanol, the filtrate was concentrated in vacuoto give methyl 6-(2,6-difluoro-3-hydroxyphenyl)-5-fluoropicolinate as agrey oil in 86% yield. LC/MS=284.0 (M+H), Rt=0.90 min.

Synthesis of methyl 6-(2,6-difluoro-3-hydroxyphenyl)picolinate

Method 7 was followed using methyl6-(3-(benzyloxy)-2,6-difluorophenyl)picolinate (1.0 equiv.) to yieldmethyl 6-(2,6-difluoro-3-hydroxyphenyl)picolinate as a light brown solidin 96% yield. LC/MS=266.0 (M+H), Rt=0.68 min.

Synthesis of methyl 6-(2-fluoro-5-formylphenyl)picolinate

To a solution of methyl 6-bromopicolinate (1.0 equiv.) in DME (0.03 M)in a microwave vial was added Pd(dppf)Cl₂-DCM (0.05 equiv.),2-fluoro-5-formylphenylboronic acid (1.5 equiv.) and 2M Na₂CO₃ (2equiv.). The reagents were heated to 120° C. for 20 min. A mixture ofthe desired product and the corresponding carboxylic acid was detectedby LC/MS, the reaction was diluted with ethyl acetate, washed with HCl(pH=5), the acidic phase was extracted with ethyl acetate, the combinedorganic layers were dried with magnesium sulfate, filtered, andconcentrated in vacuo to provide a light brown solid. The solid wasdissolved in MeOH and treated with 3 equiv. of TMS-diazomethane at roomtemperature. Upon complete conversion of the carboxylic acid to thecorresponding methyl ester, the reaction was concentrated in vacuo andthe crude material was purified via silica gel column chromatography(ISCO) eluting with 30% ethyl acetate in hexanes to provide methyl6-(2-fluoro-5-formylphenyl)picolinate as a yellow solid in 58% yield.LC/MS=260.0 (M+H), Rt=0.70 min.

Synthesis of (E)-methyl 6-(2-fluoro-5-(prop-1-enyl)phenyl)picolinate

To a solution of methyl 6-(2-fluoro-5-formylphenyl)picolinate (1.0equiv.) in MeOH (0.17 M) was added ethyltriphenylphosphonium bromide(1.0 equiv.) followed by sodium methoxide (1.5 equiv.). The reaction washeated to 65° C. for 5 hours, then cooled to room temperature andconcentrated in vacuo. The crude material was purified via silica gelcolumn chromatography (ISCO) eluting with 50% ethyl acetate in hexanesto provide (E)-methyl 6-(2-fluoro-5-(prop-1-enyl)phenyl)picolinate as awhite solid in 81% yield. LC/MS=272.0 (M+H), Rt=0.73 min.

Synthesis of methyl 6-(2-fluoro-5-propylphenyl)picolinate

To a solution of (E)-methyl6-(2-fluoro-5-(prop-1-enyl)phenyl)-picolinate (1.0 equiv.) in MeOH (0.04M) was 10% Pd/C (0.5 equiv.) and the reaction was placed under anatmosphere of hydrogen and left stirring overnight. The mixture wasfiltered over a pad of Celite and washed with MeOH. The filtrate wasconcentrated in vacuo to provide methyl6-(2-fluoro-5-propylphenyl)picolinate as a light grey oil in 97% yield.LC/MS=274.2 (M+H), Rt=0.61 min.

Synthesis of 6-(2-fluoro-5-propylphenyl)picolinic acid

To a solution of methyl 6-(2-fluoro-5-propylphenyl)picolinate (1.0equiv.) in THF was added lithium hydroxide (10 equiv.) and the reactionwas stirred at room temperature for 1 hour. The THF solvent was removedin vacuo and the remaining basic phase was acidified with concentratedHCl. The aqueous layer was extracted with ethyl acetate (2×), theorganic phase was dried with sodium sulfate, filtered and concentratedto give 6-(2-fluoro-5-propylphenyl)picolinic acid in 35% yield.LC/MS=260.2 (M+H), Rt=0.36 min.

Method 8

Synthesis of methyl6-(2,6-difluoro-3-(trifluoromethyl-sulfonyloxy)phenyl)-5-fluoropicolinate

To a solution of methyl6-(2,6-difluoro-3-hydroxyphenyl)-5-fluoropicolinate (1.0 equiv.) in DCM(0.2 M) was added DIEA (2.0 equiv.) and1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide(1.5 equiv.). The reaction was allowed to stir overnight at roomtemperature. The solution was quenched with water, the organic phase wasdried with sodium sulfate, and concentrated. The crude material waspurified via ISCO chromatography eluting with ethyl acetate and hexanes(0-30% ethyl acetate). The pure fractions were concentrated to givemethyl6-(2,6-difluoro-3-(trifluoromethylsulfonyloxy)phenyl)-5-fluoropicolinateas the desired product as a clear oil in 68% yield. LC/MS=416.1 (M+H),Rt=1.08 min.

Synthesis of methyl6-(2,6-difluoro-3-(trifluoromethylsulfonyloxy)-phenyl)picolinate

Method 8 was followed using methyl6-(2,6-difluoro-3-(trifluoromethylsulfonyloxy)phenyl)-5-fluoropicolinate(1.0 equiv.) to yield methyl6-(2,6-difluoro-3-(trifluoromethylsulfonyloxy)phenyl)picolinate as acolorless oil in >99% yield. LC/MS=397.9 (M+H), Rt=1.03 min.

Synthesis of 6-(2,6-difluoro-3-methylphenyl)-5-fluoropicolinic acid

To a solution methyl6-(2,6-difluoro-3-(trifluoromethylsulfonyloxy)phenyl)-5-fluoropicolinate(1.0 equiv.) in dioxane and water (10:1, 0.15M) was added methyl boronicacid (3.0 equiv.) and potassium carbonate (3.0 equiv.). The reaction wasdegassed with nitrogen for 10 min, then Pd(PPh₃)₄ (0.1 equiv.) was addedto the solution and heated to 100° C. for 3 hours. LC/MS of the reactionat this point indicated complete conversion to the carboxylic acidproduct (M+H=268). Cooled to room temperature and added water and ethylacetate. The two layers were separated, the aqueous phase was acidifiedwith concentrated HCl to pH=1 and extracted with ethyl acetate. Theorganic phase was dried with sodium sulfate, filtered, and concentratedunder vacuo to give 6-(2,6-difluoro-3-methylphenyl)-5-fluoropicolinicacid as a clear oil in 97% yield. LC/MS=268.1 (M+H), Rt=0.82 min.

Synthesis of methyl 6-(2,6-difluoro-3-methylphenyl)picolinate

To a solution of methyl6-(2,6-difluoro-3-(trifluoromethylsulfonyloxy)-phenyl)picolinate (1.0equiv.) in toluene was added Pd(dppf)Cl₂-DCM (0.1 equiv.) followed bydimethyl zinc (3.0 equiv.). The solution turned from orange to brightyellow. The reaction was heated to 80° C. for 2 hours at which time,LC/MS analysis indicated complete conversion to product. The reactionwas cooled to room temperature, diluted with ethyl acetate and washedwith brine. The organic layer was dried with magnesium sulfate,filtered, and concentrated in vacuo to provide methyl6-(2,6-difluoro-3-methylphenyl)picolinate as a brown oil in quantitativeyield. LC/MS=264.0 (M+H), Rt=0.90 min.

Synthesis of 6-(2,6-difluoro-3-methylphenyl)picolinic acid

To a solution of methyl 6-(2,6-difluoro-3-methylphenyl)picolinate (1.0equiv.) in THF was added sodium hydroxide (10 equiv.) and the reactionwas stirred for 2 hours. The solution was diluted with ethyl acetate andwashed with 1N NaOH (2×). The combined basic aqueous washes werecombined and acidified with concentrated HCl. The acidic aqueous phasewas extracted with ethyl acetate (2×), the combined organic layers weredried with magnesium sulfate, filtered, and concentrated in vacuo toprovide 6-(2,6-difluoro-3-methylphenyl)picolinic acid as a white solidin 85% yield. LC/MS=250.0 (M+H), Rt=0.76 min.

Synthesis of 6-(3-ethyl-2,6-difluorophenyl)picolinic acid

To a solution of methyl6-(2,6-difluoro-3-(trifluoromethylsulfonyloxy)-phenyl)picolinate (1.0equiv.) in toluene (0.15M) was added Pd(dppf)Cl₂-DCM (0.1 equiv.)followed by diethyl zinc (3.0 equiv.). The solution turned from orangeto bright yellow. The reaction was heated to 70° C. for 2 hours at whichtime, LC/MS analysis indicated a mixture of 1:3:1 ratio of hydrolyzedproduct, desired product and unknown by-product. The reaction was cooledto room temperature, diluted with ethyl acetate and washed with 1N NaOH(2×). The organic layer was dried over magnesium sulfate, filtered, andconcentrated in vacuo to provide a brown oil. The oil was redissolved inTHF and treated with 1N NaOH for one hour. The reaction was then dilutedwith ethyl acetate and washed with 1N NaOH (2×). The basic washings werecombined, acidified with concentrated HCl and extracted with ethylacetate (3×). The organic phase was dried with magnesium sulfate,filtered, and concentrated in vacuo to provide6-(3-ethyl-2,6-difluorophenyl)picolinic acid as a light brown oilin >99% yield. LC/MS=264.1 (M+H), Rt=0.88 min.

Method 9

A homogeneous solution of 1 eq each of amine, carboxylic acid, HOAT andEDC in DMF, at a concentration of 0.5 M, was left standing for 24 hoursat which time water and ethyl acetate were added. The organic phase wasdried with sodium sulfate and purified via silica gel columnchromatography eluting with ethyl acetate and hexanes to give thedesired protected amide product. Alternatively the crude reactionmixture was directly purified by HPLC. Upon lyophilization, the TFA saltof the protected amide product was obtained. Alternatively, the HPLCfractions could be added to EtOAc and solid Na₂CO₃, separated and washedwith NaCl_((sat.)). Upon drying over MgSO₄, filtering and removing thevolatiles in vacuo, the protected amide product was obtained as a freebase. Alternatively, the crude reaction mixture was used for thedeprotection step without further purification.

If an N-Boc protected amine was present, it was removed by treating withexcess 4M HCl/dioxane for 14 hours or by treating with 25% TFA/CH₂Cl₂for 2 hours. Upon removal of the volatiles in vacuo, the material waspurified by RP HPLC yielding after lyophilization the amide product asthe TFA salt. Alternatively, the HPLC fractions could be added to EtOAcand solid Na₂CO₃, separated and washed with NaCl_((sat.)). Upon dryingover MgSO₄, filtering and removing the volatiles in vacuo the free basewas obtained. Upon dissolving in MeCN/H₂O, adding 1 eq. of 1 N HCl andlyophilizing, the HCl salt of the amide product was obtained.

If an N-Boc1,2 amino alcohol cyclic carbamate was present, prior to Bocdeprotection the cyclic carbamate could be cleaved by treating withCs₂CO₃ (0.5 eq) in ethanol at a concentration of 0.1 M for three hours.After removal of volatiles in vacuo, the Boc amino group was deprotectedas described above.

If an N-Boc, OAc group were present, prior to Boc deprotection, theacetate group could be cleaved by treating with K₂CO₃ (2.0 equiv.) inethanol at a concentration of 0.1M for 24 hours.

If an N-phthalimide group was present, the amine was deprotected bytreating with hydrazine in MeOH at 65° C. for three hours. Upon coolingand filtering off the white precipitate, the filtrate was concentratedand purified by RP HPLC to yield the amino amide product.

If a TBDMS ether was present, it was deprotected prior to Boc removal bytreating with 6N HCl, THF, methanol (1:2:1) at room temperature for 12h. After removal of volatiles in vacuo, the Boc amino group wasdeprotected as described above. Alternatively, the TBDMS ether and Bocgroup could be both deprotected with 6N HCl, THF, methanol (1:2:1) ifleft at rt for 24 hours, or heated at 60° C. for 3 hours.

If a OMe group was present, it was deprotected by treating with 1M BBr₃in DCM (2.0 equiv.) for 24 hours. Water was added dropwise and thevolatiles were removed in vacuo. The material was purified via reversephase HPLC as described above.

If a OBn group was present, it was deprotected by treatment with 10%Pd/C (0.2 equiv.) under an atmosphere of hydrogen in ethyl acetate andmethanol (1:2). Upon completion, the reaction was filtered throughCelite, washed with methanol, and the filtrate was concentrated invacuo.

Synthesis of(+/−)-3-amino-N-(4-(3-amino-4-hydroxycyclohex-1-enyl)pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamide

Following Method 9, (+/−)-tert-butyl3-(3-aminopyridin-4-yl)-6-(tert-butyldimethylsilyloxy)cyclohex-2-enylcarbamateand 3-amino-6-(2,6-difluorophenyl)-picolinic acid were coupled anddeprotected to yield(+/−)-3-amino-N-(4-(3-amino-4-hydroxycyclohex-1-enyl)pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamideas the TFA salt. LCMS (m/z): 438.2 (MH⁺), LC R_(t)=2.00 min.

Synthesis of(+/−)-3-amino-N-(4-(3-amino-4-hydroxycyclohexyl)-pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamide

Following Method 9, (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-2-(tert-butyldimethylsilyloxy)cyclohexylcarbamateand 3-amino-6-(2,6-difluorophenyl)picolinic acid were coupled anddeprotected to yield(+/−)-3-amino-N-(4-(3-amino-4-hydroxycyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamideas the TFA salt in 18% yield. LCMS (m/z): 440.3 (MH⁺), LC R_(t)=2.04min.

Following the procedures of Method 9, the following compounds wereprepared:

TABLE 1 LC/MS LC/MS (M + H on (Rf on Example No. Structure UPCL) UPCL)Chemical Name 1

442.2 0.75 6-(2,6-difluorophenyl)-5- fluoro-N-(4-((1R,3S,5S)-3-hydroxy-5- methylcyclohexyl)pyridin-3- yl)picolinamide 2

474.3 0.52 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6- difluoro-4-hydroxyphenyl)-5- fluoropicolinamide3

473.3 0.55 3-amino-N-(4-((3R,4R,5S)-3- amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin- 3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 4

473.3 0.52 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluoro-4- hydroxyphenyl)-5-fluoropicolinamide 5

455.3 0.55 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-5-fluoro-6-(2-fluoro-5-hydroxyphenyl)picolinamide 6

407.2 0.52 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2-fluoro-4- hydroxyphenyl)picolinamide 7

407.2 0.53 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2-fluoro-5- hydroxyphenyl)picolinamide 8

425.2 0.54 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- hydroxyphenyl)picolinamide 9

425.2 0.53 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-4- hydroxyphenyl)picolinamide 10

453.3 0.58 N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-4- hydroxyphenyl)picolinamide 11

469.2 0.7 N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- methylphenyl)-5- fluoropicolinamide 12

451.1 0.69 N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- methylphenyl)picolinamide 13

454.2 0.58 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6- difluoro-3- methylphenyl)picolinamide 14

445.2 0.62 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-5-fluoro-6-(2,3,5- trifluorophenyl)picolinamide 15

467.5 0.63 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(3-ethyl-2,6-difluorophenyl)picolinamide 16

428.2 0.63 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-cyclohexyl- 5-fluoropicolinamide 17

467.3 0.64 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(3-ethyl-2,6-difluorophenyl)picolinamide 18

453.2 0.66 N-(4-((1R,3R,4S)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(3-ethyl-2,6- difluorophenyl)picolinamide 19

437.2 0.67 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(3-ethyl-2,6- difluorophenyl)picolinamide 20

441.2 0.64 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- methylphenyl)-5- fluoropicolinamide 21

451.2 0.7 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(3-ethyl-2,6- difluorophenyl)picolinamide 22

472.3 0.59 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6- difluoro-3-methylphenyl)-5- fluoropicolinamide23

422.3 0.53 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2- fluorophenyl)picolinamide 24

458.3 0.51 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6- difluorophenyl)-3- fluoropicolinamide 25

440.3 0.54 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-5-fluoro-6-(2- fluorophenyl)picolinamide 26

440.2 0.53 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6- difluorophenyl)picolinamide 27

455.3 0.66 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- methylphenyl)-5- fluoropicolinamide 28

423.2 0.61 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- methylphenyl)picolinamide 29

437.2 0.64 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- methylphenyl)picolinamide 30

453.2 0.59 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluoro-3-methylphenyl)picolinamide 31

453.2 0.6 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluoro-3-methylphenyl)picolinamide 32

443.2 0.55 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- hydroxyphenyl)-5- fluoropicolinamide 33

425.2 0.53 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-5- hydroxyphenyl)picolinamide 34

425.2 0.52 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-3-fluoro-6-(2-fluoro-5- hydroxyphenyl)picolinamide 35

421.1 0.56 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2-fluoro-5- hydroxyphenyl)picolinamide 36

439.2 0.57 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- hydroxyphenyl)picolinamide 37

421.1 0.56 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2-fluoro-4- hydroxyphenyl)picolinamide 38

439.2 0.57 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-4- hydroxyphenyl)picolinamide 39

439.2 0.55 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-3-fluoro-6-(2-fluoro-4- hydroxyphenyl)picolinamide 40

471.2 0.62 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluoro-3- methylphenyl)-5-fluoropicolinamide 41

439.2 0.57 N-(4-((1R,3R,4S)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- methylphenyl)picolinamide 42

457.2 0.56 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- hydroxyphenyl)-3- fluoropicolinamide 43

439.2 0.56 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-3-fluoro-6-(2-fluoro-5- hydroxyphenyl)picolinamide 44

471.2 0.62 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluoro-3- methylphenyl)-5-fluoropicolinamide 45

453.1 0.61 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-5- methoxyphenyl)picolinamide 46

439.1 0.56 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-5- hydroxyphenyl)picolinamide 47

487.1 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluoro-3- methoxyphenyl)-5-fluoropicolinamide 48

427.2 0.59 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3- yl)-6-(2,3,5-trifluorophenyl)picolinamide 49

455.2 0.63 N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 50

457.2 0.58 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 51

456.1 0.58 3-amino-N-(4-((1R,3S,5S)-3- amino-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 52

439.1 0.68 N-(4-((1S,3S,4S,5R)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6- difluorophenyl)picolinamide 53

413.3 0.55 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-5-fluoro-6-((1s,4s)-4- hydroxycyclohexyl) picolinamide 54

427.3 0.59 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-((1s,4s)-4- hydroxycyclohexyl) picolinamide 55

427.3 0.55 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-((1r,4r)-4- hydroxycyclohexyl) picolinamide 56

413.3 0.48 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-5-fluoro-6-((1r,4r)-4- hydroxycyclohexyl) picolinamide 57

423.3 0.64 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-(2- fluorophenyl)picolinamide 58

419.3 0.67 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2-fluoro-5- methylphenyl)picolinamide 59

437.3 0.67 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-5- methylphenyl)picolinamide 60

441.2 0.70 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,3-difluorophenyl)-5- fluoropicolinamide 61

441.2 0.68 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,5-difluorophenyl)-5- fluoropicolinamide 62

405.2 0.67 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2- fluorophenyl)picolinamide 63

423.2 0.65 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6- difluorophenyl)picolinamide 64

483.2 0.66 N-(4-((1R,3R,4S)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-5-fluoro-6-(2-fluoro-5- propoxyphenyl)picolinamide 65

458.1 0.55 3-amino-N-(4-((1R,3R,4S)-3- amino-4-hydroxycyclohexyl)pyridin-3- yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 66

440.1 0.55 3-amino-N-(4-((1R,3R,4S)-3- amino-4-hydroxycyclohexyl)pyridin-3- yl)-5-fluoro-6-(2-fluorophenyl)picolinamide 67

498.2 0.66 3-amino-N-(4-((1R,3R,4S)-3- amino-4-hydroxycyclohexyl)pyridin-3- yl)-5-fluoro-6-(2-fluoro-5-propoxyphenyl)picolinamide 68

480.2 0.65 3-amino-N-(4-((1R,3R,4S)-3- amino-4-hydroxycyclohexyl)pyridin-3- yl)-6-(2-fluoro-5-propoxyphenyl)picolinamide 69

441.3 0.67 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-3- fluoropicolinamide 70

441.3 0.70 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 71

441.3 0.66 N-(4-((1S,3R,5R)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-3- fluoropicolinamide 72

441.3 0.70 N-(4-((1S,3R,5R)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 73

453.1 0.7 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-5-fluoro-6-(2-fluoro-5-methylphenyl)picolinamide 74

435.0 0.6 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2-fluoro-5- methylphenyl)picolinamide75

439.2 0.57 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-5-fluoro-6-(2- fluorophenyl)picolinamide76

439.2 0.55 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6- difluorophenyl)picolinamide 77

441.3 0.62 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,4-difluorophenyl)-5- fluoropicolinamide 78

425.2 0.52 N-(4-((1R,3R,4S)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(2,6- difluorophenyl)picolinamide 79

427.2 0.58 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,3-difluorophenyl)-5- fluoropicolinamide 80

427.2 0.54 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-3- fluoropicolinamide 81

427.2 0.57 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,5-difluorophenyl)-5- fluoropicolinamide 82

427.2 0.58 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,4-difluorophenyl)-5- fluoropicolinamide 83

407.1 0.51 N-(4-((1R,3R,4S)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(2- fluorophenyl)picolinamide 84

465.2 0.62 N-(4-((1R,3R,4S)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(2-fluoro-5- propoxyphenyl)picolinamide 85

457.2 0.56 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 86

457.0 0.56 N-(4-((1R,3R,4S,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 87

425.1 0.52 N-(4-((1R,3R,4S)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-5-fluoro-6-(2- fluorophenyl)picolinamide 88

443.0 0.53 N-(4-((1S,3S,4R)-3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 89

427.3 0.63 N-(4-(3-amino-4-hydroxy-5- methylcyclohexyl)pyridin-3-yl)-6-cyclohexyl-5- fluoropicolinamide 90

424.3 0.6 3-amino-N-(4-(3-amino-4- hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-cyclohexylpicolinamide 91

411.3 0.67 N-(4-(3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-cyclohexyl-5- fluoropicolinamide 92

438.3 0.51 3-amino-N-(4-(trans)-3- amino-4-hydroxycyclohex-1-enyl)pyridin-3-yl)-6-(2,6- difluorophenyl)picolinamide 93

438.3 0.51 3-amino-N-(4-(cis)-3-amino- 4-hydroxycyclohex-1-enyl)pyridin-3-yl)-6-(2,6- difluorophenyl)picolinamide 94

454.1 0.54 3-amino-N-(4-(3-amino-4- hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6- difluorophenyl)picolinamide 95

454.3 0.54 3-amino-N-(4-(3-amino-4- hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6- difluorophenyl)picolinamide 96

458.1 0.54 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6- difluorophenyl)-5- fluoropicolinamide 97

454.1 0.55 3-amino-N-(4-(3-amino-4- hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6- difluorophenyl)picolinamide 98

454.1 0.54 3-amino-N-(4-(3-amino-4- hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6- difluorophenyl)picolinamide 99

427.2 0.55 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 100

442.2 0.59 3-amino-N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 101

482.2 0.56 2-amino-4-(3-(3-amino-6- (2,6- difluorophenyl)picolinamido)pyridin-4-yl)cyclohexyl acetate 102

440.3 0.52 3-amino-N-(4-(3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(2,6- difluorophenyl)picolinamide 103

455.3 0.53 3-amino-N-(4-((3R,4S,5R)-3- amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin- 3-yl)-6-(2,6- difluorophenyl)picolinamide104

440.2 0.52 3-amino-N-(4-((1R,3S,4S)-3- amino-4-hydroxycyclohexyl)pyridin-3- yl)-6-(2,6- difluorophenyl)picolinamide 105

438.2 2.92 3-amino-6-(2,6- difluorophenyl)-N-(4-(3-hydroxy-5-methylcyclohex-1- enyl)pyridin-3-yl)picolinamide 106

394.3 0.74 3-amino-N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-cyclohexylpicolinamide 107

421.9 0.59 3-amino-N-(4-(3- aminocyclohex-1- enyl)pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamide 108

473.3 0.67 N-(4-((1R,3R,5S)-3-amino-5- (trifluoromethyl)cyclohexyl)pyridin-3-yl)-6-(2-fluoro-5- methylphenyl)picolinamide 109

507.2 0.65 N-(4-((1R,3R,5S)-3-amino-5- (trifluoromethyl)cyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- methoxyphenyl)picolinamide 110

510.2 0.64 3-amino-N-(4-((1R,3R,5S)-3- amino-5-(trifluoromethyl)cyclohexyl) pyridin-3-yl)-6-(2,6- difluorophenyl)-5-fluoropicolinamide 111

465.3 0.72 N-(4-((1R,3R,5S)-3-amino-5- (trifluoromethyl)cyclohexyl)pyridin-3-yl)-6-cyclohexyl-5- fluoropicolinamide 112

492.2 0.62 3-amino-N-(4-((1R,3R,5S)-3- amino-5-(trifluoromethyl)cyclohexyl) pyridin-3-yl)-6-(2,6-difluorophenyl)picolinamide 113

458.2 0.55 N-(4-((3S,4S,5R)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6- difluorophenyl)-5- fluoropicolinamide 114

441.1 0.61 N-(4-((3R,4R,5S)-3,4- dihydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-5-fluoro-6- (2-fluorophenyl)picolinamide 115

441.1 0.6 6-(2,6-difluorophenyl)-N-(4- ((3R,4R,5S)-3,4-dihydroxy-5-methylpiperidin-1-yl)pyridin- 3-yl)picolinamide 116

459.1 0.61 6-(2,6-difluorophenyl)-N-(4- ((3R,4R,5S)-3,4-dihydroxy-5-methylpiperidin-1-yl)pyridin- 3-yl)-5-fluoropicolinamide 117

458.2 0.62 6-(2,6-Difluorophenyl)-N-(4- ((1R,3R,4R,5S)-3,4- dihydroxy-5-methylcyclohexyl)pyridin-3- yl)-5-fluoropicolinamide 118

423.2 0.32 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-3-fluoro-2,3′- bipyridine-6-carboxamide 119

441.2 0.46 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-3,3′-difluoro- 2,4′-bipyridine-6- carboxamide 120

423.1 0.3 N-(4-((3R,4R,5S)-3-amino-4- hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-3-fluoro-2,4′- bipyridine-6-carboxamide 121

473.1 0.68 3-amino-6-(2,6- difluorophenyl)-N-(4- ((1R,3R,4R,5S)-3,4-dihydroxy-5- methylcyclohexyl)pyridin-3- yl)-5-fluoropicolinamide

Synthesis of6-bromo-N-(4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)pyridin-3-yl)-5-fluoropicolinamide

Following Method9,4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)pyridin-3-amineand 6-bromo-5-fluoropicolinic acid were coupled and following additionof EtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,6-bromo-N-(4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)pyridin-3-yl)-5-fluoropicolinamidewas obtained. LCMS (m/z): 455.3 (MH⁺); LC R_(t)=2.09 min.

Synthesis of6-bromo-N-(4-((1R,3S)-3-(1,3-dioxoisoindolin-2-yl)-cyclohexyl)pyridin-3-yl)-5-fluoropicolinamide

Following Method9,2-(3-(3-aminopyridin-4-yl)cyclohexyl)isoindoline-1,3-dione and6-bromo-5-fluoropicolinic acid were coupled and following addition ofEtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,6-bromo-N-(4-((1R,3S)-3-(1,3-dioxoisoindolin-2-yl)cyclohexyl)pyridin-3-yl)-5-fluoropicolinamidewas obtained. LCMS (m/z): 523.2/525.2 (MH⁺); LC R_(t)=3.31 min.

Synthesis of3-amino-6-bromo-N-(4-((1R,3S)-3-(1,3-dioxoisoindolin-2-yl)cyclohexyl)pyridin-3-yl)-5-fluoropicolinamide

Following Method9,2-(3-(3-aminopyridin-4-yl)cyclohexyl)-isoindoline-1,3-dione and3-amino-6-bromo-5-fluoropicolinic acid were coupled and followingaddition of EtOAc and washing with H₂O, NaCl_((sat.)) and drying overMgSO₄,3-amino-6-bromo-N-(4-((1R,3S)-3-(1,3-dioxoisoindolin-2-yl)cyclohexyl)pyridin-3-yl)-5-fluoropicolinamidewas obtained. LCMS (m/z): 538.1/540.1 (MH⁺); LC R_(t)=3.46 min.

Synthesis of tert-butyl(1S,3R,5S)-3-(3-(6-bromo-5-fluoropicolinamido)-pyridin-4-yl)-5-methylcyclohexylcarbamate

Following Method 9, tert-butyl(1S,3R,5S)-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate and6-bromo-5-fluoropicolinic acid were coupled and following addition ofEtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,tert-butyl(1S,3R,5S)-3-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-5-methylcyclohexylcarbamatewas obtained. LCMS (m/z): 507.1/509.1 (MH⁺), R_(t)=0.90 min.

Synthesis of(1R,2R,4R,6S)-4-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexylacetate

Following Method 9,(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexylacetate and 6-bromo-5-fluoropicolinic acid were coupled and followingaddition of EtOAc and washing with H₂O, NaCl_((sat.)) and drying overMgSO₄,(1R,2R,4R,6S)-4-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexylacetate was obtained. LCMS (m/z): 567.2 (MH⁺), R_(t)=0.82 min.

Synthesis of (+/−)-tert-butyl5-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate

Following Method 9, (+/−)-tert-butyl5-(3-aminopyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylateand 6-bromo-5-fluoropicolinic acid were coupled and following additionof EtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,(+/−)-tert-butyl5-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-7-methyl-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylatewas obtained. LCMS (m/z): 549.2/551.2 (MH⁺), R_(t)=0.78 min.

Synthesis of tert-butyl5-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate

Following Method 9, tert-butyl5-(3-aminopyridin-4-yl)-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylateand 6-bromo-5-fluoropicolinic acid were coupled and following additionof EtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,tert-butyl5-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylatewas obtained. LCMS (m/z): 537.1 (MH⁺); LCMS R_(t)=0.71 min.

Synthesis of6-bromo-N-(4-((1R,5R)-5-(1,3-dioxoisoindolin-2-yl)-3,3-dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide

Following Method9,2-((1R,5R)-5-(3-aminopyridin-4-yl)-3,3-dimethylcyclohexyl)isoindoline-1,3-dioneand 6-bromo-5-fluoropicolinic acid were coupled and following additionof EtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,6-bromo-N-(4-((1R,5R)-5-(1,3-dioxoisoindolin-2-yl)-3,3-dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamidewas obtained. LCMS (m/z): 551/553 (MH⁺), R_(t)=0.95 min.

Synthesis ofN-(4-((1R,3R,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-yl)-6-bromo-5-fluoropicolinamide

Following Method 9,4-((1R,3R,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amineand 6-bromo-5-fluoropicolinic acid were coupled and following additionof EtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,N-(4-((1R,3R,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-yl)-6-bromo-5-fluoropicolinamidewas obtained. LCMS (m/z): 652.5, 652.4 (MH⁺); LC R_(t)=5.82 min.

Synthesis ofN-(4-((1S,3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-yl)-6-bromo-5-fluoropicolinamide

Following Method 9,4-((1S,3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amineand 6-bromo-5-fluoropicolinic acid were coupled and following additionof EtOAc and washing with H₂O, NaCl_((sat.)) and drying over MgSO₄,N-(4-((1S,3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-yl)-6-bromo-5-fluoropicolinamidewas obtained. LCMS (m/z): 652.5, 652.4 (MH⁺); LC R_(t)=5.83 min.

Method 10

Synthesis of6-(2,6-difluorophenyl)-5-fluoro-N-(4-(3-hydroxy-5-methylcyclohex-1-enyl)pyridin-3-yl)picolinamide

A solution of6-bromo-N-(4-(3-(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-enyl)pyridin-3-yl)-5-fluoropicolinamide(1.0 equiv), 2,6-difluorophenyl boronic acid (3.0 equiv.),tetrakistriphenylphosphine (0.2 equiv.) and triethylamine (3.0 equiv.)in 1:1 EtOH/toluene (0.1 M) was heated at 120° C. with microwaveirradiation for 1200 seconds. Upon cooling, removal of the volatiles invacuo, the Suzuki product was directly purified by reverse phase HPLC.The product fraction was lyophilized and the resulting TBDMS ether wasdeprotected as described in Method 9 yielding, after RP HPLCpurification and lyophilization,6-(2,6-difluorophenyl)-5-fluoro-N-(4-(3-hydroxy-5-methylcyclohex-1-enyl)pyridin-3-yl)picolinamideas the TFA salt. LCMS (m/z): 438.2 (MH⁺); LC Rt=2.00 min.

Synthesis ofN-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide

A solution of6-bromo-N-(4-((1R,3S)-3-(1,3-dioxoisoindolin-2-yl)-cyclohexyl)pyridin-3-yl)-5-fluoropicolinamide(1.0 equiv), 2,6-difluorophenyl boronic acid (3.0 equiv.),tetrakistriphenylphosphine (0.2 equiv.) and triethylamine (3.0 equiv.)in 1:1 EtOH/toluene (0.1 M) was heated at 120° C. with microwaveirradiation for 1200 seconds. Upon cooling, removal of the volatiles invacuo, the Suzuki product was directly purified by reverse phase HPLC.The product fraction was lyophilized and the resulting phthalimide groupwas deprotected as described in Method 9 yielding, after RP HPLCpurification and lyophilization,N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamideas the TFA salt. LCMS (m/z): 427.2 (MH⁺); LC Rt=2.26 min.

Synthesis of3-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide

A solution of3-amino-6-bromo-N-(4-((1R,3S)-3-(1,3-dioxoisoindolin-2-yl)cyclohexyl)pyridin-3-yl)-5-fluoropicolinamide(1.0 equiv), 2,6-difluorophenyl boronic acid (3.0 equiv.),tetrakistriphenylphosphine (0.2 equiv.) and triethylamine (3.0 equiv.)in 1:1 EtOH/toluene (0.1 M) was heated at 120° C. with microwaveirradiation for 1200 seconds. Upon cooling, removal of the volatiles invacuo, the Suzuki product was directly purified by reverse phase HPLC.The product fraction was lyophilized and the resulting phthalimide groupwas deprotected as described in Method 9 yielding, after RP HPLCpurification and lyophilization,3-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamideasthe TFA salt. LCMS (m/z): 442.2 (MH⁺); LC Rt=2.24 min.

Synthesis ofN-(4-(3-amino-4-hydroxycyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide

A solution of tert-butyl5-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-2-oxohexahydrobenzo[d]oxazole-3(2H)-carboxylate(1.0 equiv), 2,6-difluorophenyl boronic acid (3.0 equiv.),tetrakistriphenylphosphine (0.2 equiv.) and triethylamine (3.0 equiv.)in 1:1 EtOH/toluene (0.1 M) was heated at 120° C. with microwaveirradiation for 1200 seconds. Upon cooling, removal of the volatiles invacuo, the Suzuki product was directly purified by reverse phase HPLC.The product fraction was lyophilized and the resulting cyclic carbamateand Boc groups were deprotected as described in Method 9 yielding, afterRP HPLC purification and lyophilization,N-(4-(3-amino-4-hydroxycyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamideasthe TFA salt. LCMS (m/z): 443.2 (MH⁺); LC Rt=2.11 min.

Synthesis of5-amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-3,3′-difluoro-2,4′-bipyridine-6-carboxamide

A solution of3-amino-6-bromo-N-(4-((1R,3S)-3-(1,3-dioxoisoindolin-2-yl)cyclohexyl)pyridin-3-yl)-5-fluoropicolinamide(1.0 equiv), 3-fluoropyridin-4-yl-boronic acid (3.0 equiv.),tetrakistriphenylphosphine (0.2 equiv.) and triethylamine (3.0 equiv.)in 1:1 EtOH/toluene (0.1 M) was heated at 120° C. with microwaveirradiation for 1200 seconds. Upon cooling, removal of the volatiles invacuo, the Suzuki product was directly purified by reverse phase HPLC.The product fraction was lyophilized and the resulting phthalimide groupwas deprotected as described in Method 9 yielding, after RP HPLCpurification and lyophilization,5-amino-N-(4-((1R,3S)-3-aminocyclohexyl)-pyridin-3-yl)-3,3′-difluoro-2,4′-bipyridine-6-carboxamideas the TFA salt. LCMS (m/z): 425.1 (MH⁺); LC Rt=2.08 min.

Synthesis ofN-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3-hydroxyphenyl)-5-fluoropicolinamide

To a solution of tert-butyl(1S,3R,5S)-3-(3-(6-bromo-5-fluoropicolinamido)pyridin-4-yl)-5-methylcyclohexylcarbamate(1.0 equiv.) in a microwave vial was added2,6-difluoro-3-hydroxyphenylboronic acid (5.0 equiv.), KF (5.5 equiv.)and Pd₂(dba)₃ (0.2 equiv.) followed by THF and water (10:1, 0.03 M). Tothis mixture was added P(t-Bu)₃ (0.4 equiv.) and the reaction was heatedin the microwave at 100° C. for 30 min. The organic phase was thenseparated, the aqueous layer was washed with ethyl acetate, and theorganics were combined and concentrated in vacuo. The crude mixture waspurified via prep-HPLC, the product fractions were lyophilized and theresulting BOC group was deprotected as described in Method 9 yielding,after RP HPLC purification and lyophilization,N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3-hydroxyphenyl)-5-fluoropicolinamideas the TFA salt. LCMS (m/z): 457.2 (MH⁺); LC Rt=2.17 min.

The following compounds were prepared using Method 10:

TABLE 2 LC/MS LC/MS Example No./ (M + H on (Rf on NVP ID Structure UPCL)UPCL) Chemical Name 122

471.2 0.58 N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- hydroxyphenyl)-5- fluoropicolinamide 123

485.2 0.65 N-(4-((1R,5R)-5-amino-3,3- dimethylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- methoxyphenyl)-5- fluoropicolinamide 124

443.2 0.54 N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- hydroxyphenyl)-5- fluoropicolinamide 125

457.2 0.57 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-3- hydroxyphenyl)-5- fluoropicolinamide 126

507.1 0.65 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-5-fluoro-6-(2-fluoro-5-(trifluoromethyl)phenyl) picolinamide 127

507.1 0.65 N-(4-((1R,3R,4R,5S)-3- amino-4-hydroxy-5-methylcyclohexyl)pyridin-3- yl)-5-fluoro-6-(2-fluoro-3-(trifluoromethyl)phenyl) picolinamide 128

457.2 0.58 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluoro-4- hydroxyphenyl)-5- fluoropicolinamide 129

459.2 0.62 N-(4-((1R,3S,5S)-3-amino-5- methylcyclohexyl)pyridin-3-yl)-5-fluoro-6-(2,3,6- trifluorophenyl)picolinamide 130

472.1 0.56 3-amino-N-(4-(3-amino-4- hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 131

458.0 0.57 3-amino-N-(4-(3-amino-4- hydroxycyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 132

472.1 0.56 3-amino-N-(4-(3-amino-4- hydroxy-5-methylcyclohexyl)pyridin-3- yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide 133

456.3 0.60 3-amino-N-(4-(3-amino-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 134

457.1 0.57 N-(4-(-3-amino-4-hydroxy-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 135

457.1 0.55 N-(4-(3-amino-4-hydroxy-5- methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5- fluoropicolinamide 136

425.1 0.46 5-amino-N-(4-((1R,3S)-3- aminocyclohexyl)pyridin-3-yl)-3,3′-difluoro-2,4′- bipyridine-6-carboxamide 137

440.2 0.75 6-(2,6-difluorophenyl)-5- fluoro-N-(4-(3-hydroxy-5-methylcyclohex-1- enyl)pyridin-3- yl)picolinamide 138

458.2 0.64 6-(2,6-difluorophenyl)-N-(4- ((1R,3R,4S,5R)-3,4- dihydroxy-5-methylcyclohexyl)pyridin-3- yl)-5-fluoropicolinamide

Example 139 Synthesis ofN-(4-((1R,3R,4S,5S)-3-amino-4-fluoro-5-methylcyclohexyl)-pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide

To a solution of tert-butyl(1R,2R,3S,5R)-5-(3-(6-(2,6-difluorophenyl)-5-fluoropicolinamido)pyridin-4-yl)-2-hydroxy-3-methylcyclohexylcarbamate(1.0 equiv.) in DCM (0.04 M) at 0° C. was added DAST (1.0 equiv.). Thereaction was stirred for 1.5 h at 0° C., then TFA (10 equiv.) was addedto the reaction. After 2 h, the reaction was concentrated in vacuo andthe residue was purified via prep-HPLC to affordN-(4-((1R,3R,4S,5S)-3-amino-4-fluoro-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamideas the TFA salt. LCMS (m/z): 459.3 (MH⁺); LC Rt=2.39 min.

In addition to LC/MS and LC characterization, representative compoundswere analyzed by ¹H-NMR. The following are typical spectra of thecompounds of the invention.

Example # ¹H-NMR data 50 HCl salt, 1H-NMR (400 MHz, DMSO-d6): d 10.54(bs, 1H), 8.80 (bs, 1H), 8.55 (d, 1H), 8.34 (dd, 1H), 8.20 (t, 1H), 8.00(bs, 2H), 7.69 (m, 1H), 7.56 (d, 1H), 7.34 (t, 2H), 3.10-3.0 (m, 2H),2.83 (m, 1H), 2.03 (d, 1H), 1.76-1.59 (m, 2H), 1.40 (m, 1H), 1.31-1.21(m, 1H), 0.92 (d, 3H). 52 HCl salt, 1H-NMR (DMSO-d6): d 10.4 (s, 1H),8.71 (s, 1H), 8.37 (d, 1H), 8.14-8.20 (m, 2H), 7.94 (bs, 2H), 7.86-7.88(m, 2H), 7.54-7.58 (m, 1H), 7.30 (d, 1H), 7.22-7.26 (m, 2H), 2.09-3.02(m, 2H), 2.78 (m, 1H), 1.96-1.99 (m, 1H), 1.68-1.71 (m, 1H), 1.60 (q,1H), 1.37 (m, 1H), 1.15-1.24 (m, 1H), 0.88 (d, 3H) 70 HCl salt, 1H NMR(400 MHz, DMSO-d6): d 10.59 (s, 1H), 8.92 (s, 1H), 8.62 (d, 1H), 8.37(dd, 1H), 8.23 (t, 1H), 8.19 (bs, 2H), 7.68-7.71 (m, 2H), 7.36-7.40 (m,2H), 3.01-3.10 (m, 2H), 2.01-2.05 (m, 1H), 1.94-1.97 (m, 1H), 1.72-1.76(m, 1H), 1.46-1.53 (m, 2H), 1.01-1.13 (m, 2H), 0.89 (d, 3H) 85 HCl salt,1H NMR (DMSO-d6): d 10.37 (s, 1H), 8.61 (s, 1H), 8.41 (d, 1H), 8.29 (dd,1H), 8.13 (t, 1H), 7.8 (bs, 2H), 7.69-7.61 (m, 1H), 7.34-7.28 (m, 3H),3.061 (m, 1H), 2.86 (m, 1H), 1.76-1.63 (m, 2H), 1.53-1.47 (m, 1H),1.4-1.34 (m, 2H), 0.82 (d, 3H). 88 HCl salt, 1H NMR (DMSO-d6): d 10.42(s, 1H), 8.62 (s, 1H), 8.46 (m, 1H), 8.32 (m, 1H), 8.18 (t, 1H), 7.76(m, 2H), 7.67 (m, 1H), 7.35 (m, 3H), 5.33 (brs, 1H), 3.108 (m, 2H), 2.88(m, 2H), 1.65 (m, 2H), 1.48 (m, 3H). 96 HCl salt, 1H NMR (400 MHz,CD3OD): d 9.09 (s, 1 H), 8.46 (dd, 1 H), 8.39 (dd, 1 H), 8.05 (t, 1 H),7.57-7.67 (m, 1 H), 7.53 (d, 1 H), 7.16-7.25 (m, 2 H), 4.03-4.12 (m, 1H), 3.85-3.94 (m, 1 H), 3.20 (s, 3 H), 2.70-2.80 (m, 1 H), 1.67-1.79 (m,1 H), 0.83 (d, 3 H). 99 free-base, 1H NMR (CDCl3): d 9.93 (s, 1H), 9.38(s, 1H), 8.40-8.45 (m, 1H), 8.40 (d, 1H), 7.74-7.80 (m, 1H), 7.47-7.55(m, 1H), 7.19 (d, 1H), 7.06-7.13 (m, 2H), 2.68-2.83 (m, 2H), 1.97-2.05(m, 1H), 1.65-1.95 (m, 5H), 1.22-1.40 (m, 3H), 1.04-1.15 (m, 1H). 100HCl salt, 1H NMR (DMSO-d6): d 10.13 (s, 1H), 8.82 (s, 1H), 8.41 (d, 1H),7.94 (bs, 2H), 7.52-7.62 (m, 1H), 7.36 (d, 1H), 7.36 (bs, 2H), 7.20-7.31(m, 3H), 2.78-2.88 (m, 2H), 1.70-2.02 (m, 4H), 1.16-1.54 (m, 4H). 102HCl salt, 1H NMR (400 MHz, DMSO-d6): d 10.59 (s, 1H), 9.30 (s, 1H), 8.54(d, 1H) 8.08 (br s, 3H), 7.75 (d, 1H), 7.65 (d, 1H), 7.60-7.56 (m, 1H),7.49 (d, 1H), 7.33 (t, 2H), 4.04 (br s, 1H), 3.16 (br s, 2H) 3.05 (br t,1H), 1.98-1.20 (m, 7H) 116 HCl salt, 1H-NMR (400, d6-DMSO): d 10.47 (s,1H), 8.56 (s, 1H), 8.33 (dd, 1H), 8.26 (dd, 1H), 8.20 (t, 1H), 7.62-7.72(m, 1H), 7.30-7.35 (m, 3H), 3.82-3.92 (m, 2H), 3.18-3.22 (m, 1H),2.84-2.91 (m, 1H), 2.69 (t, J = 13.2, 1H), 1.38-1.46 (m, 1H), 0.69 (d,3H). 128 HCl salt, 1H-NMR (400, d6-DMSO): d 11.00 (s, 1H), 10.46 (s,1H), 8.55 (d, 1H), 8.29 (dd, 1H), 8.15 (t, 1H), 8.05 (bs, 2H), 7.54 (d,1H), 6.72 (d, 2H), 3.04-3.10 (m, 1H), 2.92-3.04 (m, 1H), 2.01 (d, 1H),1.95 (d, 1H), 1.74 (d, 1H), 1.42-1.52 (m, 2H), 0.97-1.08 (m, 2 H), 0.88(d, 3H).

Example 140

Pim1 ATP Depletion Assay

The activity of PIM1 is measured using a luciferase-luciferin based ATPdetection reagent to quantify ATP depletion resulting fromkinase-catalyzed phosphoryl transfer to a peptide substrate. Compoundsto be tested are dissolved in 100% DMSO and directly distributed intowhite 384-well plates at 0.5 μl per well. To start the reaction, 10 μlof 5 nM Pim1 kinase and 80 μM BAD peptide (RSRHSSYPAGT-OH) in assaybuffer (50 mM HEPES pH 7.5, 5 mM MgCl₂, 1 mM DTT, 0.05% BSA) is addedinto each well. After 15 minutes, 10 μl of 40 μM ATP in assay buffer isadded. Final assay concentrations are 2.5 nM PIM1, 20 μM ATP, 40 μM BADpeptide and 2.5% DMSO. The reaction is performed until approximately 50%of the ATP is depleted, then stopped with the addition of 20 μlKinaseGlo Plus (Promega Corporation) solution. The stopped reaction isincubated for 10 minutes and the remaining ATP detected via luminescenceon the Victor2 (Perkin Elmer). Compounds of the foregoing examples weretested by the Pim1 ATP depletion assay and found to exhibit an IC₅₀values as shown in Table 3, below. IC₅₀, the half maximal inhibitoryconcentration, represents the concentration of a test compound that isrequired for 50% inhibition of its target in vitro.

Example 141

Pim2 ATP depletion assay

The activity of PIM2 is measured using a luciferase-luciferin based ATPdetection reagent to quantify ATP depletion resulting fromkinase-catalyzed phosphoryl transfer to a peptide substrate. Compoundsto be tested are dissolved in 100% DMSO and directly distributed intowhite 384-well plates at 0.5 μl per well. To start the reaction, 10 μlof 10 nM Pim2 kinase and 20 μM BAD peptide (RSRHSSYPAGT-OH) in assaybuffer (50 mM HEPES pH 7.5, 5 mM MgCl₂, 1 mM DTT, 0.05% BSA) is addedinto each well. After 15 minutes, 10 μl of 8 μM ATP in assay buffer isadded. Final assay concentrations are 5 nM PIM2, 4 μM ATP, 10 μM BADpeptide and 2.5% DMSO. The reaction is performed until approximately 50%of the ATP is depleted, then stopped with the addition of 20 μlKinaseGlo Plus (Promega Corporation) solution. The stopped reaction isincubated for 10 minutes and the remaining ATP detected via luminescenceon the Victor2 (Perkin Elmer). Compounds of the foregoing examples weretested by the Pim2 ATP depletion assay and found to exhibit an IC₅₀values as shown in Table 3, below.

Example 142

Pim3 ATP Depletion Assay

The activity of PIM3 is measured using a luciferase-luciferin based ATPdetection reagent to quantify ATP depletion resulting fromkinase-catalyzed phosphoryl transfer to a peptide substrate. Compoundsto be tested are dissolved in 100% DMSO and directly distributed intowhite 384-well plates at 0.5 μl per well. To start the reaction, 10 μlof 10 nM Pim3 kinase and 200 μM BAD peptide (RSRHSSYPAGT-OH) in assaybuffer (50 mM HEPES pH 7.5, 5 mM MgCl₂, 1 mM DTT, 0.05% BSA) is addedinto each well. After 15 minutes, 10 μl of 80 μM ATP in assay buffer isadded. Final assay concentrations are 5 nM PIM1, 40 μM ATP, 100 μM BADpeptide and 2.5% DMSO. The reaction is performed until approximately 50%of the ATP is depleted, then stopped by the addition of 20 μl KinaseGloPlus (Promega Corporation) solution. The stopped reaction is incubatedfor 10 minutes and the remaining ATP detected via luminescence on theVictor2 (Perkin Elmer). Compounds of the foregoing examples were testedby the Pim3 ATP depletion assay and found to exhibit an IC₅₀ values asshown in Table 3, below.

Example 143

Cell Proliferation Assay

KMS11 (human myeloma cell line), were cultured in IMDM supplemented with10% FBS, sodium pyruvate and antibiotics. Cells were plated in the samemedium at a density of 2000 cells per well into 96 well tissue cultureplates, with outside wells vacant, on the day of assay. MMl.s (humanmyeloma cell line), were cultured in RPMI1640 supplemented with 10% FBS,sodium pyruvate and antibiotics. Cells were plated in the same medium ata density of 5000 cells per well into 96 well tissue culture plates,with outside wells vacant, on the day of assay.

Test compounds supplied in DMSO were diluted into DMSO at 500 times thedesired final concentrations before dilution into culture media to 2times final concentrations. Equal volumes of 2× compounds were added tothe cells in 96 well plates and incubated at 37° C. for 3 days.

After 3 days plates were equilibrated to room temperature and equalvolume of CellTiter-Glow Reagent (Promega) was added to the culturewells. The plates were agitated briefly and luminescent signal wasmeasured with luminometer. The percent inhibition of the signal seen incells treated with DMSO alone vs. cells treated with control compoundwas calculated and used to determine EC₅₀ values (i.e., theconcentration of a test compound that is required to obtain 50% of themaximum effect in the cells) for tested compounds, as shown in Table 3.

Using the procedures of Examples 140 (Pim1 ATP depletion assay), 141(Pim2 ATP depletion assay), and 142 (Pim3 ATP depletion assay), the IC₅₀concentration of compounds of the previous examples were determined asshown in the following table 3.

Using the procedures of Example 143 (cell proliferation assay), the EC₅₀concentration of compounds of the examples in were determined in KMS11cells as shown in Table 3.

TABLE 3 KMS11- Pim2 Iuc Example No./ Pim1 IC50 Pim3 EC50 NVP IDStructure IC50 μM μM IC50 μM μM  1

0.001 0.018 0.006 7.6  2

0.001 0.001 0.001 0.07  3

0.001 0.001 0.001 0.01  4

0.001 0.003 0.002 1.3  5

0.003 0.020 0.009 4.8  6

0.002 0.012 0.003 4.1  7

0.001 0.008 0.002 0.33  8

0.001 0.004 0.002 0.51  9

0.001 0.008 0.002 1.6  10

0.001 0.012 0.006 2.9  11

0.001 0.005 0.004 2.6  12

0.001 0.010 0.004 2.4  13

0.001 0.004 0.003 0.67  14

0.006 0.040 0.012 8.5  15

0.003 0.027 0.006 5.5  16

0.001 0.003 0.003 1.7  17

0.001 0.013 0.005 3.5  18

0.003 0.062 0.007 6.3  19

0.003 0.054 0.007 4.5  20

0.001 0.007 0.003 1.5  21

0.002 0.013 0.006 3.3  22

0.001 0.002 0.003 0.21  23

0.002 0.005 0.003 1.9  24

0.001 0.002 0.001 0.62  25

0.001 0.002 0.002 0.37  26

0.001 0.002 0.002 0.29  27

0.001 0.003 0.002 0.95  28

0.001 0.011 0.002 2.2  29

0.001 0.004 0.003 1.4  30

0.002 0.012 0.004 2.1  31

0.002 0.007 0.004 1.1  32

0.001 0.004 0.003 0.39  33

0.001 0.009 0.003 1.4  34

0.004 0.067 0.006 6.0  35

0.001 0.006 0.003 0.67  36

0.001 0.003 0.003 0.24  37

0.002 0.007 0.005 1.5  38

0.001 0.004 0.003 0.73  39

0.007 0.028 0.012 6.6  40

0.001 0.003 0.002 0.99  41

0.002 0.027 0.005 2.0  42

0.001 0.002 0.002 3.0  43

0.001 0.006 0.002 2.2  44

0.001 0.002 0.002 1.9  45

0.001 0.002 0.002 1.3  46

0.001 0.002 0.002 0.76  47

0.001 0.004 0.003 1.3  48

0.007 0.076 0.009  49

0.001 0.003 0.002 1.8  50

0.001 0.003 0.002 1.2  51

0.001 0.002 0.001 0.31  52

0.001 0.003 0.002 0.83  53

0.010 0.149 0.065 >10  54

0.003 0.026 0.024 3.8  55

0.003 0.011 0.030 4.9  56

0.011 0.081 0.102 >10  57

0.001 0.004 0.002 1.4  58

0.001 0.008 0.003 1.8  59

0.001 0.003 0.002 1.1  60

0.001 0.005 0.003 1.3  61

0.001 0.006 0.004 1.2  62

0.001 0.007 0.003 2.4  63

0.001 0.003 0.002 0.53  64

0.002 0.076 0.005 5.6  65

0.001 0.004 0.003 0.16  66

0.001 0.004 0.002 0.52  67

0.001 0.007 0.003 1.2  68

0.001 0.008 0.004 1.4  69

0.003 0.007 0.006 1.0  70

0.001 0.002 0.002 0.48  71

0.662 1.947 1.05  72

0.095 0.522 0.369  73

0.001 0.008 0.004 1.4  74

0.001 0.017 0.004 2.9  75

0.001 0.008 0.003 2.1  76

0.001 0.003 0.002 0.83  77

0.001 0.013 0.003 3.9  78

0.002 0.015 0.003 3.6  79

0.002 0.020 0.003 4.6  80

0.006 0.044 0.007 7.9  81

0.002 0.025 0.005 5.940  82

0.003 0.080 0.009 >10  83

0.004 0.048 0.005 >10  84

0.004 0.163 0.007 6.9  85

0.001 0.003 0.002 0.41  86

0.031 0.124 0.106 6.1  87

0.002 0.035 0.005 7.7  88

0.001 0.011 0.005 0.79  89

0.008 0.021 0.029  90

0.003 0.010 0.012  91

0.003 0.012 0.021 2.9  92

0.002 0.009 0.005  93

0.001 0.008 0.005  94

0.001 0.003 0.005 0.239  95

0.001 0.005 0.006 0.537  96

0.001 0.001 0.001 0.03  97

0.002 0.010 0.007 3.3  98

0.002 0.005 0.005 0.81  99

0.001 0.005 0.003 0.93 100

0.001 0.001 0.001 0.28 101

0.002 0.007 0.005 102

0.001 0.004 0.004 0.87 103

0.002 0.008 0.004 104

0.003 0.013 0.005 105

0.001 0.006 0.004 106

0.005 0.022 0.014 107

0.002 0.007 0.006 0.93 108

0.001 0.007 0.003 109

0.001 0.002 0.002 110

0.001 0.001 0.001 111

0.002 0.005 0.011 112

0.001 0.001 0.001 113

0.004 0.089 0.028 114

0.001 0.003 0.001 0.64 115

0.001 0.002 0.001 0.59 116

0.001 0.001 0.001 0.29 117

0.001 0.006 0.002 2.5 118

0.004 0.029 0.009 4.6 119

0.001 0.004 0.002 1.2 120

0.003 0.018 0.007 8.5 121

0.001 0.001 0.002 122

0.001 0.002 0.002 0.23 123

0.001 0.004 0.002 0.49 124

0.001 0.002 0.001 0.78 125

0.001 0.001 0.001 0.41 126

0.002 0.068 0.017 127

0.011 0.131 0.027 128

0.001 0.001 0.001 0.26 129

0.001 0.003 0.002 1.2 130

0.002 0.007 0.008 1.0 131

0.002 0.007 0.006 0.43 132

0.001 0.005 0.006 0.38 133

0.001 0.004 0.007 0.24 134

0.002 0.016 0.008 1.9 135

0.023 0.088 0.036 136

0.002 0.024 0.009 6.4 137

0.001 0.020 0.007 138

0.002 0.045 0.006 139

0.001 0.005 0.004 1.9

Example 144

Biological Method Pharmacology Target Modulation and Efficacy Study inMultiple Myeloma Xenograft Model

KMS11-luc multiple myeloma cancer cells, obtained from Suzanne Trudel(University Health Network, Toronto, Canada), express stable luciferaseachieved by retroviral transfection and were maintained in DMEMsupplemented with 10% heat-inactivated fetal bovine serum with 1%glutamine (Invitrogen, Inc.). Female SCID/bg mice (8-12 weeks old, 20-25g, Charles River) were used for all in vivo pharmacology studies. Themice were housed and maintained in accordance with state and federalguidelines for the humane treatment and care of laboratory animals, andreceived food and water ad libitum. Cancer cells were harvested frommid-log phase cultures, viable cell count was established with anautomated cell counter (Vi-CELL, Beckman-Coulter), and cells wereresuspended in equal parts HBSS and Matrigel (Invitrogen, Inc.). Tenmillions cells were subcutaneously injected into the right flank of eachmouse. Compound treatment was initiated when tumor size reached 250-350mm³ for PK/PD studies, and 150-250 mm³ for efficacy studies, with tumorvolumes determined using StudyDirector software (StudyLog Systems,Inc.). All compound treatment was administered orally.

For in vivo target modulation in PK/PD time-course studies,tumor-bearing mice were administered a single oral dose of vehicle orcompound at different concentrations. At 1, 8 and 24 hours after dosing,tumor tissues and blood samples were taken from individual mice.Resected tumor tissues were snap frozen and pulverized using a liquidnitrogen-cooled cryomortar and pestle. Blood samples were taken bycardiac puncture, and plasma was separated utilizing centrifugationtubes containing lithium heparin and plasma separator (BD Microtainer).Frozen tumor samples were lysed in cold buffer (Meso Scale Discovery)supplemented with EDTA free protease inhibitor (Roche), phosphataseinhibitors 1 and 2, and 1M NaF (Sigma) according to manufacturer'sinstructions. Following homogenization with a dounce apparatus or byMagNA Lyser (Roche), clear supernatant was obtained followingcentrifugation at 300×g for 30 minutes at 4° C. and proteinconcentration was determined by BCA (BioRad). Target modulation wasdetermined using the Meso Scale phospho-Bad^(Ser112)/total Bad duplexkit, according to manufacturer's instructions. Briefly, an equal amountof protein was loaded into each well of a Meso Scalephospho-Serine¹¹²/total Bad duplex 96-well plate (Meso Scale Discovery)and samples were incubated for 30 minutes at room temperature orovernight at 4° C., shaking. Plates were washed with 1×MSD wash buffer,and Sulfo-Tag detection antibody was added to the wells and incubatedfor 1 hour at room temperature, shaking. The plates were washed againand captured analyte detected following the addition of Read Buffer T tothe wells. Plates were read on a SECTOR Imager 6000 Instrument (MesoScale Discovery). Ratios of the signal from pBad to total Bad were usedto correct for variability between samples. Data shown in the followingtable express the percent inhibition of pBad^(Ser112) phosphorylationrelative to total Bad phosphorylation by representative compounds of theinvention, normalized to vehicle control group. The extent of modulationis expressed as a percent, relative to vehicle control (n.d., notdetermined).

Compound of Example No. 1 hr 8 hr 24 hr 99 (50 mg/kg) 40 55 0 99 (100mg/kg) 62 66 24 70 (25 mg/kg) 34 50 n.d. 70 (50 mg/kg) 28 62 0 70 (100mg/kg) 5 67 68 96 (25 mg/kg) 0 24 n.d. 96 (50 mg/kg) 44 69 16 96 (100mg/kg) 58 71 53

For efficacy studies, tumor-bearing mice were randomized into groupswith equivalent tumor volume variation ranging from 150-250 mm³utilizing the StudyDirector software (StudyLog Systems, Inc.). Followingrandomization, mice were dosed orally daily or twice daily at multiplecompound concentrations in 200 μl incipient. Tumor growth and animalbody weight was measured at least twice weekly, and daily clinicalobservations were used to monitor potential toxicities related to thetreatment. Animals were removed from study if tumor volume exceeded 2500mm³, or if body weight loss exceeded 20% of initial measurements.

Efficacy of the compound of Example 99 was evaluated in the KMS11-lucxenograft model, with mice receiving oral administration of the compoundof Example 99 twice daily at 50 and 100 mg/kg, and once daily at 100mg/kg for 14 days. Dosing was initiated when tumor sizes reachedapproximately 250 mm³. As shown in FIG. 1, the compound of Example 99exhibited dose-dependant effects in vivo, with tumor growth inhibitionobserved with 50 mg/kg twice daily (92%) and 100 mg/kg twice daily (4%regression). Once daily administration of 100 mg/kg was less efficacious(65%) than when dosed twice daily. These results correlate with theextent and magnitude of pBad^(Ser112) modulation, and suggest thatextensive and prolonged target modulation is required for maximumefficacy.

Efficacy of the compound of Example 70 was evaluated in the KMS11-lucxenograft model, with mice receiving oral administration of the compoundof Example 80 twice daily at 25 and 50 mg/kg, and once daily at 100mg/kg for 14 days. Dosing was initiated when tumor sizes reachedapproximately 225 mm³. As shown in FIG. 2, the compound of Example 70exhibited dose-dependant effects in vivo, with tumor growth inhibitionobserved for 25 mg/kg (65%) and 50 mg/kg (100%). Significant tumorgrowth inhibition was also observed for 100 mg/kg once daily (84%).

Efficacy of the compound of Example 96 was evaluated in the KMS11-lucxenograft model, with mice receiving oral administration of the compoundof Example 96 twice daily at 25 and 50 mg/kg, and once daily at 100mg/kg for 14 days. Dosing was initiated when tumor sizes reachedapproximately 225 mm³. As shown in FIG. 3, compound the compound ofExample 96 exhibited dose-dependant effects in vivo, with tumor growthinhibition observed for 25 mg/kg (67%), and 50 mg/kg (96%). Significanttumor growth inhibition was also observed for 100 mg/kg once daily(88%).

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

The invention claimed is:
 1. A method for treating multiple myeloma,wherein the method comprises the step of: administering to a patient inneed of such treatment an effective amount ofN-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamideof Formula or a pharmaceutically acceptable salt thereof,


2. The method of claim 1, whereinN-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamideis combined with at least one pharmaceutically acceptable carrier.